TW201730212A

TW201730212A - ROR1-binding molecules, and methods of use thereof

Info

Publication number: TW201730212A
Application number: TW106104961A
Authority: TW
Inventors: 巴斯娃緹巴拉特; 萊斯利強生; 保羅摩爾; 佛曼安德森瑞夫; 愛利歐波維尼
Original assignee: 宏觀基因股份有限公司
Priority date: 2016-02-17
Filing date: 2017-02-15
Publication date: 2017-09-01
Also published as: US20170233472A1; UY37127A; WO2017142928A1; AR108034A1

Abstract

The present invention is directed to optimized ROR1-binding molecules having enhanced affinity and superior ability to mediate redirected cytotoxicity of tumor cells relative to prior ROR1-binding molecules. More specifically, the invention relates to optimized ROR1-binding molecules that comprise Variable Light Chain and/or Variable Heavy Chain (VH) Domains that have been optimized for binding to an epitope present on the human ROR1 polypeptide so as to exhibit enhanced binding affinity for human ROR1 and/or a reduced immunogenicity upon administration to recipient subjects. The invention particularly pertains to bispecific, trispecific or multispecific ROR1-binding molecules, including bispecific diabodies, BiTEs, bispecific antibodies, trivalent binding molecules, etc. that comprise: (i) such optimized ROR1-binding Variable Domains and (ii) a domain capable of binding to an epitope of a molecule present on the surface of an effector cell. The invention is also directed to pharmaceutical compositions that contain any of such ROR1-binding molecules, and to methods involving the use of any of such ROR1-binding molecules in the treatment of cancer and other diseases and conditions.

Description

ROR1-結合分子及其使用方法ROR1-binding molecule and method of use thereof

[相關申請的交叉引用][Cross-reference to related applications]

本申請要求美國專利申請系列號62/296,267 (2016年2月17日提交；待決)的優先權，該申請以其整體併入本文。The present application claims priority to U.S. Patent Application Serial No. 62/296,267, filed on Feb. 17, s.

[序列表的参考][Reference to Sequence Listing]

根據37 C.F.R. 1.821以及下面的條款，本申請包括一個或多個序列表，其以電腦-可讀介質(檔案名：1301_0139PCT_ST25.txt，2017年1月11日創建，並且大小為159,339位元組)公開，該檔通過引用以其整體併入本文。In accordance with 37 CFR 1.821 and the following clauses, this application includes one or more sequence listings on a computer-readable medium (file name: 1301_0139PCT_ST25.txt, created on January 11, 2017, and having a size of 159,339 bytes) The disclosure is hereby incorporated by reference in its entirety.

本發明涉及優化的ROR1-結合分子，其相對於現有的ROR1-結合分子具有增強的親和力和介導對腫瘤細胞的重定向細胞毒性的更好的能力。更具體地，本發明涉及包括可變輕鏈和/或可變重鏈(VH)結構域的優化的ROR1-結合分子，所述可變輕鏈和/或可變重鏈(VH)結構域已經被優化以結合存在於人ROR1多肽上的表位元，以便顯示出對人ROR1增強的結合親和力和/或在施用於接受受試者後降低的免疫原性。本發明特別涉及雙特異性，三特異性或多特異性ROR1-結合分子，包括雙特異性雙抗體、BiTEs、雙特異性抗體、三價結合分子等，其包括：(i)這樣的優化的結合ROR1的可變結構域和(ii)能夠結合存在於效應細胞表面上的分子的表位元的結構域。本發明還涉及含有任何這類ROR1-結合分子的藥物組合物，以及涉及包括在癌症和其它疾病和狀況的治療中使用任何這類ROR1-結合分子的方法。The present invention relates to an optimized ROR1-binding molecule that has enhanced affinity relative to existing ROR1-binding molecules and a better ability to mediate redirected cytotoxicity to tumor cells. More specifically, the invention relates to an optimized ROR1-binding molecule comprising a variable light chain and/or a variable heavy chain (VH) domain, said variable light chain and/or variable heavy chain (VH) domain It has been optimized to bind to epitopes present on human ROR1 polypeptides in order to exhibit enhanced binding affinity for human ROR1 and/or reduced immunogenicity upon administration to a subject. The invention particularly relates to bispecific, trispecific or multispecific ROR1-binding molecules, including bispecific diabodies, BiTEs, bispecific antibodies, trivalent binding molecules, etc., which comprise: (i) such optimized Binding to the variable domain of ROR1 and (ii) the domain of an epitope capable of binding to a molecule present on the surface of an effector cell. The invention also relates to pharmaceutical compositions containing any such ROR1-binding molecules, as well as to methods of using any such ROR1-binding molecules in the treatment of cancer and other diseases and conditions.

受體酪氨酸激酶樣孤兒受體1(“ROR1”)是屬於細胞表面受體的ROR亞家族的I型膜蛋白(Masiakowski, P.等(1992). “A Novel Family Of Cell Surface Receptors With Tyrosine Kinase-Like Domain ,” J. Biol. Chem. 267:26181-26190)。 ROR1是在胚胎發育過程中由許多組織表達的癌-胚(onco-embryonic)抗原，其在大多數成熟組織中不存在(Paganoni, S.等(2005) “Neurite Extension In Central Neurons: A Novel Role For The Receptor Tyrosine Kinases ROR1 And ROR2 ,” J. Cell Sci. 118:433-446)，並且在許多血液和實體惡性腫瘤包括卵巢、結腸、肺、淋巴瘤、皮膚、胰腺、睾丸、膀胱、子宮、***、腎上腺、乳腺、B-細胞惡性腫瘤以及一些癌幹細胞中表達(Zhang, S.等(2012) “The Onco-Embryonic Antigen ROR1 Is Expressed by a Variety of Human Cancers, ” Am. J. Pathol. 6:1903-1910；Zhang, S.等(2012) “ROR1 Is Expressed In Human Breast Cancer And Associated With Enhanced Tumor-Cell Growth ,” PLoS One 7:e31127；Daneshmanesh, A.H.等(2008) “ROR1, A Cell Surface Receptor Tyrosine Kinase Is Expressed In Chronic Lymphocytic Leukemia And May Serve As A Putative Target For Therapy ,” Int. J. Cancer 123:1190-1195；Zhang, S.等(2014) “Ovarian Cancer Stem Cells Express ROR1, Which Can Be Targeted For Anti-Cancer-Stem-Cell Therapy ,” Proc. Natl. Acad. Sci. (U.S.A.) 111:17266-71)。ROR1表達與顯示較低分化形態的高級腫瘤有關，以及與不良的臨床結果相關(Zhang, S.等(2012) “The Onco-Embryonic Antigen ROR1 Is Expressed by a Variety of Human Cancers, ” Am. J. Pathol. 6:1903-1910；Zhang, H.等(2014) “ROR1 Expression Correlated With Poor Clinical Outcome In Human Ovarian Cancer ,” Sci. Rep. 4:5811,第1-7頁)。The receptor tyrosine kinase-like orphan receptor 1 ("ROR1") is a type I membrane protein of the ROR subfamily belonging to cell surface receptors (Masiakowski, P. et al. (1992). " A Novel Family Of Cell Surface Receptors With Tyrosine Kinase-Like Domain ," J. Biol. Chem. 267: 26181-26190). ROR1 is an onco-embryonic antigen expressed by many tissues during embryonic development, which does not exist in most mature tissues (Paganoni, S. et al. (2005) “ Nerite Extension In Central Neurons: A Novel Role For The Receptor Tyrosine Kinases ROR1 And ROR2 ," J. Cell Sci. 118: 433-446), and in many blood and solid malignancies including ovary, colon, lung, lymphoma, skin, pancreas, testis, bladder, uterus, Expression in prostate, adrenal gland, breast, B-cell malignancies, and some cancer stem cells (Zhang, S. et al. (2012) “ The Onco-Embryonic Antigen ROR1 Is Expressed by a Variety of Human Cancers, ” Am. J. Pathol. 6 :1903-1910; Zhang, S. et al. (2012) “ ROR1 Is Expressed In Human Breast Cancer And Associated With Enhanced Tumor-Cell Growth ,” PLoS One 7:e31127; Daneshmanesh, AH et al. (2008) “ ROR1, A Cell Surface Receptor Tyrosine Kinase Is Expressed In Chronic Lymphocytic Leukemia And May Serve As A Putative Target For Therapy ,” Int. J. Cancer 123: 1190-1195; Zhang, S. et al. (2014) “ Ovar Ian Cancer Stem Cells Express ROR1, Which Can Be Targeted For Anti-Cancer-Stem-Cell Therapy ," Proc. Natl. Acad. Sci. (USA) 111:17266-71). ROR1 expression is associated with advanced tumors showing lower differentiated morphology and associated with poor clinical outcomes (Zhang, S. et al. (2012) “ The Onco-Embryonic Antigen ROR1 Is Expressed by a Variety of Human Cancers, ” Am. J. Pathol. 6:1903-1910; Zhang, H. et al. (2014) " ROR1 Expression Correlated With Poor Clinical Outcome In Human Ovarian Cancer ," Sci. Rep. 4:5811, pp. 1-7).

鑒於ROR1癌-胚抗原的受限制的表達，已經探索了許多針對靶ROR1的不同的基於免疫的策略，包括抗體、抗體藥物綴合物、表達T細胞的嵌合抗原受體(CAR)和靶向ROR1的納米顆粒(Choi, M.Y.,等(2015) “Pre-clinical Specificity and Safety of UC-961, a First-In-Class Monoclonal Antibody Targeting ROR1 ,” Clin Lymphoma Myeloma Leuk 15(Suppl):S167-S169；Daneshmanesh, A.H.,等(2012) “Monoclonal Antibodies Against ROR1 Induce Apoptosis Of Chronic Lymphocytic Leukemia (CLL) cells ,” Leukemia 26:1348-1355；Yang, J.等(2011) “Therapeutic Potential And Challenges Of Targeting Receptor Tyrosine Kinase ROR1 With Monoclonal Antibodies In B-Cell Malignancies ,” PLoS One 6:e21018；Baskar, S.等(2012) “Targeting Malignant B Cells With An Immunotoxin Against ROR1, ” MAbs 4:349-361；Berger, C.等(2015) “Safety Of Targeting ROR1 In Primates With Chimeric Antigen Receptor-Modified T Cells ,” Cancer Immunol. Res. 3:206-216；Hudecek, M.等(2010) “The B-Cell Tumor-Associated Antigen ROR1 Can Be Targeted With T Cells Modified To Express A ROR1-Specific Chimeric Antigen Receptor ,” Blood. 116:4532-4541；Mani, R.等(2015) “Tumor Antigen ROR1 Targeted Drug Delivery Mediated Selective Leukemic But Not Normal B-Cell Cytotoxicity In Chronic Lymphocytic Leukemia ,” Leukemia 29:346-355)。In view of the restricted expression of ROR1 cancer-embryo antigens, a number of different immunological-based strategies for targeting ROR1 have been explored, including antibodies, antibody drug conjugates, chimeric antigen receptors (CARs) expressing T cells, and targets. Nanoparticles to ROR1 (Choi, MY, et al. (2015) “ Pre-clinical Specificity and Safety of UC-961, a First-In-Class Monoclonal Antibody Targeting ROR1 ,” Clin Lymphoma Myeloma Leuk 15 (Suppl): S167-S169 Daneshmanesh, AH, et al. (2012) " Monoclonal Antibodies Against ROR1 Induce Apoptosis Of Chronic Lymphocytic Leukemia (CLL) cells ," Leukemia 26: 1348-1355; Yang, J. et al. (2011) " Therapeutic Potential And Challenges Of Targeting Receptor Tyrosine Kinase ROR1 With Monoclonal Antibodies In B-Cell Malignancies ,” PLoS One 6:e21018; Baskar, S. et al. (2012) “ Targeting Malignant B Cells With An Immunotoxin Against ROR1, ” MAbs 4:349-361; Berger, C. et al. . (2015) "Safety Of Targeting ROR1 In Primates With Chimeric Antigen Receptor-Modified T Cells," Cancer Immunol Res 3: 206-216; Hudecek, M.. (2010) "The B-Cell Tumor-Associated Antigen ROR1 Can Be Targeted With T Cells Modified To Express A ROR1-Specific Chimeric Antigen Receptor," Blood 116:. 4532-4541; Mani, R. et (2015) "Tumor Antigen ROR1 Targeted Drug Delivery Mediated Selective Leukemic But Not Normal B-Cell Cytotoxicity In Chronic Lymphocytic Leukemia ,” Leukemia 29: 346-355).

然而，儘管所有現有技術的進步，仍然需要具有增強的抗腫瘤活性和/或降低的免疫原性的高親和力的ROR1-結合分子。本發明滿足了這種需要和對於癌症的改進療法的需要。However, despite all advances in the art, there is still a need for high affinity ROR1-binding molecules with enhanced anti-tumor activity and/or reduced immunogenicity. The present invention addresses this need and the need for improved therapies for cancer.

詳細地，本發明提供了這樣的優化的ROR1-結合分子，其包括可變輕鏈結構域和可變重鏈結構域，其中所述可變輕鏈結構域具有SEQ ID NO:8 :的氨基酸序列(CDR_L 殘基以底線表示)： QLVLTQSPSASASLGX₁ SVX₂ LTC TLSSGHKT DTID WYQQQPGKAPRYLMX₃ LEGSGSYNKGS GVPDRFX₄ SGX₅ SSGADX₆ YLTISSLQSEDEADYYC GTDX₇ PGNYL FGGGTQLTVLG 其中X₆ 為W，並且其中： (a)X₁ 為S或G，X₂ 為K、I或N，X₃ 為K或N，X₄ 為G或不存在，X₅ 為S或I，X₇ 為Y或N； (b)X₁ 為S，X₂ 為K，X₃ 為K，X₄ 為G或不存在，X₅ 為S，並且X₇ 為N； (c)X₁ 為S，X₂ 為K，X₃ 為K，X₄ 為G或不存在，X₅ 為I，並且X₇ 為Y； (d)X₁ 為S，X₂ 為K，X₃ 為K，X₄ 為G或不存在，X₅ 為I，並且X₇ 為N；或 (e)X₁ 為S，X₂ 為K，X₃ 為K，X₄ 為G或不存在，X₅ 為S，並且X₇ 為Y。In particular, the invention provides an optimized ROR1-binding molecule comprising a variable light chain domain and a variable heavy chain domain, wherein the variable light chain domain has the amino acid of SEQ ID NO:8 : Sequence (CDR _L residues are indicated by the bottom line): QLVLTQSPSASASLG X ₁ SV X ₂ LTC TLSSGHKT DTID WYQQQPGKAPRYLM X ₃ LEGSGSYNKGS GVPDRF X ₄ SG X ₅ SSGAD X ₆ YLTISSLQSEDEADYYC GTDX ₇ PGNYL FGGGTQLTVLG where X ₆ is W, and where: (a) X ₁ is S or G, X ₂ is K, I or N, X ₃ is K or N, X ₄ is G or absent, X ₅ is S or I, X ₇ is Y or N; (b) X ₁ Is S, X ₂ is K, X ₃ is K, X ₄ is G or absent, X ₅ is S, and X ₇ is N; (c) X ₁ is S, X ₂ is K, and X ₃ is K, X ₄ is G or absent, X ₅ is I, and X ₇ is Y; (d) X ₁ is S, X ₂ is K, X ₃ is K, X ₄ is G or absent, and X ₅ is I, And X ₇ is N; or (e) X ₁ is S, X ₂ is K, X ₃ is K, X ₄ is G or absent, X ₅ is S, and X ₇ is Y.

本發明還提供了包括可變輕鏈結構域和可變重鏈結構域的優化的ROR1-結合分子，其中所述可變重鏈結構域具有SEQ ID NO:9 :的氨基酸序列(CDR_H 殘基以底線表示)： QEQLVESGGGLVQPGGSLRLSCAASGFTFS DYYMS WX₁ RQAPGKGLEWVAT IYPSSGKTYYADSX₂ KG RX₃ TISSDNAKX₄ SLYLQMNSLRAEDTAVYYCX₅ R DSYADDAALFDI WGQGTTVTVSS 其中： (a)X₁ 為V或I，X₂ 為V或A，X₃ 為L，X₄ 為N、D或Y，並且X₅ 為A或T； (b)X₁ 為V或I，X₂ 為V或A，X₃ 為F或L，X₄ 為D或Y，並且X₅ 為A或T； (c)X₁ 為V或I，X₂ 為V或A，X₃ 為F或L，X₄ 為N、D或Y，並且X₅ 為T； (d)X₁ 為V或I，X₂ 為V或A，X₃ 為L，X₄ 為N，並且X₅ 為A； (e)X₁ 為V或I，X₂ 為V或A，X₃ 為F，X₄ 為D，並且X₅ 為A； (f)X₁ 為V或I，X₂ 為V或A，X₃ 為F，X₄ 為N，並且X₅ 為T； (g)X₁ 為V或I，X₂ 為V或A，X₃ 為L，X₄ 為D，並且X₅ 為T； (h)X₁ 為I，X₂ 為A，X₃ 為F或L，X₄ 為N、D或Y，並且 X₅ 為A或T； (i)X₁ 為I，X₂ 為A，X₃ 為F，X₄ 為N，並且X₅ 為A； (j)X₁ 為I，X₂ 為A，X₃ 為L，X₄ 為N，並且X₅ 為A； (k)X₁ 為I，X₂ 為A，X₃ 為F，X₄ 為D，並且X₅ 為A； (l)X₁ 為I，X₂ 為A，X₃ 為F，X₄ 為N，並且X₅ 為T；或 (m)X₁ 為I，X₂ 為A，X₃ 為L，X₄ 為D，並且X₅ 為T。The present invention further provides ROR1- binding molecule comprises a light chain variable domain and optimized heavy chain variable domain, wherein the heavy chain variable domain having SEQ ID NO: 9: amino acid sequence (CDR _H residues The base is represented by the bottom line): QEQLVESGGGLVQPGGSLRLSCAASGFTFS DYYMS W X ₁ RQAPGKGLEWVAT IYPSSGKTYYADSX ₂ KG R X ₃ TISSDNAK X ₄ SLYLQMNSLRAEDTAVYYC X ₅ R DSYADDAALFDI WGQGTTVTVSS Where: (a) X ₁ is V or I, X ₂ is V or A, X ₃ is L X ₄ is N, D or Y, and X ₅ is A or T; (b) X ₁ is V or I, X ₂ is V or A, X ₃ is F or L, X ₄ is D or Y, and X ₅ is A or T; (c) X ₁ is V or I, X ₂ is V or A, X ₃ is F or L, X ₄ is N, D or Y, and X ₅ is T; (d) X ₁ is V or I, X ₂ is V or A, X ₃ is L, X ₄ is N, and X ₅ is A; (e) X ₁ is V or I, X ₂ is V or A, and X ₃ is F X ₄ is D, and X ₅ is A; (f) X ₁ is V or I, X ₂ is V or A, X ₃ is F, X ₄ is N, and X ₅ is T; (g) X ₁ Is V or I, X ₂ is V or A, X ₃ is L, X ₄ is D, and X ₅ is T; (h) X ₁ is I, X ₂ is A, X ₃ is F or L, X ₄ Is N, D or Y, and X ₅ is A or T; (i) X ₁ is I, X ₂ is A, X ₃ is F, X ₄ is N, and X ₅ is A; (j) X ₁ is I, X ₂ is A, X ₃ is L, X ₄ is N, and X ₅ is A; k) X ₁ is I, X ₂ is A, X ₃ is F, X ₄ is D, and X ₅ is A; (l) X ₁ is I, X ₂ is A, X ₃ is F, and X ₄ is N And X ₅ is T; or (m) X ₁ is I, X ₂ is A, X ₃ is L, X ₄ is D, and X ₅ is T.

本發明進一步涉及這類ROR1-結合分子的實施方案，其中： (a) 這類分子的VL結構域包括SEQ ID NO:11 、SEQ ID NO:20 、 SEQ ID NO:21 、SEQ ID NO:22 或SEQ ID NO:23 的氨基酸序列；和 (b) 這類分子的VH結構域包括SEQ ID NO:24 、 SEQ ID NO:25 、SEQ ID NO:26 、SEQ ID NO:30 、SEQ ID NO:31 或SEQ ID NO:32 的氨基酸序列。The invention further relates to embodiments of such ROR1-binding molecules, wherein: (a) the VL domain of such a molecule comprises SEQ ID NO: 11 , SEQ ID NO: 20 , SEQ ID NO: 21 , SEQ ID NO: 22. Or the amino acid sequence of SEQ ID NO: 23 ; and (b) the VH domain of such a molecule comprises SEQ ID NO: 24 , SEQ ID NO: 25 , SEQ ID NO: 26 , SEQ ID NO: 30 , SEQ ID NO: 31 or the amino acid sequence of SEQ ID NO:32 .

本發明進一步涉及這類ROR1-結合分子的實施方案，其中所述分子是抗體或其表位-結合片段。在本發明中，還涉及這類ROR1-結合分子的實施方案，其中所述分子是雙特異性抗體或雙抗體，特別是雙抗體或雙抗體複合物，其包括兩條、三條、四條或五條多肽鏈，每條多肽鏈各具有N-末端和C-末端，其中這些多肽鏈通過一個或多個共價鍵，特別是一個或多個共價二硫鍵締合在一起。本發明另外涉及這樣的ROR1-結合分子的實施方案，其中所述分子是三價結合分子，尤其地，其中所述三價結合分子是包括三條、四條、五條或更多條多肽鏈的共價結合的複合物。本發明還涉及這類ROR1-結合分子的實施方案，其中所述分子包括Fc區域。本發明另外涉及這類ROR1-結合分子的實施方案，其中所述分子是雙抗體並且包括白蛋白-結合結構域，尤其是去免疫化的白蛋白-結合結構域。The invention further relates to embodiments of such ROR1-binding molecules, wherein the molecule is an antibody or an epitope-binding fragment thereof. In the present invention, there is also an embodiment of such a ROR1-binding molecule, wherein the molecule is a bispecific antibody or a diabody, in particular a diabody or diabody complex, comprising two, three, four or five A polypeptide chain, each having a N-terminus and a C-terminus, wherein the polypeptide chains are associated together by one or more covalent bonds, particularly one or more covalent disulfide bonds. The invention further relates to embodiments of such ROR1-binding molecules, wherein the molecule is a trivalent binding molecule, in particular wherein the trivalent binding molecule is a covalent comprising three, four, five or more polypeptide chains Combined complex. The invention also relates to embodiments of such ROR1-binding molecules, wherein the molecule comprises an Fc region. The invention further relates to embodiments of such ROR1-binding molecules, wherein the molecule is a diabody and comprises an albumin-binding domain, in particular a deimmunized albumin-binding domain.

本發明進一步涉及另外包括Fc區域的所有這類ROR1-結合分子的實施方案，尤其地，其中所述Fc區域是包括一個或多個氨基酸修飾的變異的Fc區域，所述氨基酸修飾降低變異Fc區域對FcγR的親和力和/或增強ROR1-結合分子的血清半衰期，更具體地，其中所述修飾包括選自由以下組成的組的至少一個取代： (a) L234A； (b) L235A； (c) L234A和L235A； (d) M252Y；M252Y和S254T； (e) M252Y和T256E； (f) M252Y、S254T和T256E；和 (g) K288D和H435K；其中編號是如Kabat中的EU索引的編號。The invention further relates to embodiments of all such ROR1-binding molecules additionally comprising an Fc region, in particular wherein the Fc region is a variant Fc region comprising one or more amino acid modifications, the amino acid modification reducing the variant Fc region Affinity to FcγR and/or enhanced serum half-life of the ROR1-binding molecule, more specifically wherein the modification comprises at least one substitution selected from the group consisting of: (a) L234A; (b) L235A; (c) L234A And L235A; (d) M252Y; M252Y and S254T; (e) M252Y and T256E; (f) M252Y, S254T and T256E; and (g) K288D and H435K; where the number is the number of the EU index as in Kabat.

本發明進一步涉及這類ROR1-結合分子的實施方案，其中所述分子是雙特異性的，並且尤其涉及這樣的實施方案：其中所述分子包括能夠與ROR1的表位免疫特異性結合的兩個表位-結合位點和能夠與存在於效應細胞表面上的分子的表位免疫特異性結合的兩個表位-結合位點，或這樣的實施方案：其中所述分子包括能夠與ROR1的表位免疫特異性結合的一個表位-結合位點和能夠與存在於效應細胞表面上的分子的表位免疫特異性結合的一個表位-結合位點。The invention further relates to embodiments of such ROR1-binding molecules, wherein the molecules are bispecific, and in particular to embodiments wherein the molecule comprises two immunologically specific bindings to the epitope of ROR1 An epitope-binding site and two epitope-binding sites capable of immunospecifically binding to an epitope present on a surface of an effector cell, or an embodiment wherein the molecule comprises a table capable of interacting with ROR1 An epitope-binding site that immunospecifically binds and an epitope-binding site capable of immunospecifically binding to an epitope present on a surface of an effector cell.

本發明另外涉及這類ROR1-結合分子的實施方案，其中所述分子是三價結合分子，尤其涉及這樣的實施方案：其中所述分子包括能夠與ROR1的表位免疫特異性結合的一個表位-結合位點、能夠與存在於效應細胞表面上的第一分子的表位免疫特異性結合的一個表位-結合位點；和能夠與存在於效應細胞表面上的第二分子的表位免疫特異性結合的一個表位-結合位點，其中所述第一和第二分子不是ROR1。The invention further relates to embodiments of such ROR1-binding molecules, wherein the molecule is a trivalent binding molecule, and in particular to an embodiment wherein the molecule comprises an epitope capable of immunospecific binding to an epitope of ROR1 a binding site, an epitope-binding site capable of immunospecifically binding to an epitope of a first molecule present on the surface of an effector cell; and an epitope capable of immunizing with a second molecule present on the surface of the effector cell An epitope-binding site that specifically binds, wherein the first and second molecules are not ROR1.

本發明進一步涉及這類ROR1-結合分子的實施方案，其中所述分子能夠同時結合至ROR1和結合至第二表位，尤其涉及其中所述第二表位是存在於效應細胞表面上的第二分子的表位的實施方案(尤其是其中第二表位是CD2、CD3、CD8、CD16、TCR或NKG2D的表位，並且最特別地，其中第二表位是CD3的表位)。本發明另外涉及這類ROR1-結合分子的實施方案，其中效應細胞是細胞毒性T-細胞或自然殺傷(NK)細胞。本發明另外涉及這類ROR1-結合分子的實施方案，其中所述分子還能夠結合第三表位，尤其涉及其中第三表位是CD8的表位的實施方案。本發明還涉及這樣的分子的實施方案，其中分子介導表達ROR1的細胞和細胞毒性T細胞的協調結合。The invention further relates to embodiments of such ROR1-binding molecules, wherein the molecule is capable of binding to both ROR1 and to a second epitope, particularly where the second epitope is a second present on the surface of an effector cell An embodiment of an epitope of a molecule (especially where the second epitope is an epitope of CD2, CD3, CD8, CD16, TCR or NKG2D, and most particularly wherein the second epitope is an epitope of CD3). The invention further relates to embodiments of such ROR1-binding molecules wherein the effector cells are cytotoxic T-cells or natural killer (NK) cells. The invention further relates to embodiments of such ROR1-binding molecules, wherein the molecule is also capable of binding to a third epitope, particularly to embodiments in which the third epitope is an epitope of CD8. The invention also relates to embodiments of molecules in which the molecule mediates coordinated binding of cells expressing ROR1 and cytotoxic T cells.

本發明進一步涉及這類ROR1-結合分子的實施方案，其中所述分子包括第一多肽鏈、第二多肽鏈和第三多肽鏈，並且其中： (a) 所述第一多肽鏈包括SEQ ID NO:98 、 SEQ ID NO:101 或SEQ ID NO:102 ； (b) 所述第二多肽鏈包括SEQ ID NO:99 、 SEQ ID NO:103 或SEQ ID NO:104 ；並且 (c) 所述第三多肽鏈包括SEQ ID NO:100 。The invention further relates to embodiments of such ROR1-binding molecules, wherein the molecule comprises a first polypeptide chain, a second polypeptide chain and a third polypeptide chain, and wherein: (a) the first polypeptide chain Included in SEQ ID NO: 98 , SEQ ID NO: 101 or SEQ ID NO: 102 ; (b) the second polypeptide chain comprises SEQ ID NO: 99 , SEQ ID NO: 103 or SEQ ID NO: 104 ; c) the third polypeptide chain comprises SEQ ID NO: 100 .

本發明進一步提供了包括有效量的任何上述ROR1-結合分子和藥學上可接受的載體、賦形劑或稀釋劑的藥物組合物。The invention further provides a pharmaceutical composition comprising an effective amount of any of the above ROR1-binding molecules and a pharmaceutically acceptable carrier, excipient or diluent.

本發明另外涉及任何上述ROR1-結合分子在治療與ROR1的表達相關或以ROR1的表達為特徵的疾病或狀況中的用途，或涉及任何上述ROR1-結合分子在治療以ROR1的表達為特徵的疾病或狀況的方法中的用途，尤其地，其中與ROR1的表達相關或以ROR1的表達為特徵的疾病或狀況是癌症，更具體地，其中所述癌症選自：腎上腺腫瘤、AIDS相關的癌症、軟組織腺泡狀肉瘤、星形細胞瘤、腎上腺癌、膀胱癌、骨癌、腦和脊髓癌、轉移性腦腫瘤、B細胞癌、乳腺癌、頸動脈體瘤、宮頸癌、軟骨肉瘤、脊索瘤、嫌色細胞腎細胞癌、透明細胞癌、結腸癌、結直腸癌、皮膚良性纖維組織細胞瘤、促結締組織增生小圓細胞瘤、室管膜細胞瘤、尤文氏瘤、骨外黏液樣軟骨肉瘤、不完全性骨纖維生成、骨的纖維發育異常、膽囊癌或膽管癌、胃癌、妊娠滋養層疾病、生殖細胞瘤、頭頸癌、肝細胞癌、胰島細胞腫瘤、卡波西氏肉瘤、腎癌、白血病、脂肪肉瘤/惡性的脂肪瘤、肝癌、淋巴瘤、肺癌、成神經管細胞瘤、黑素瘤、腦膜瘤、多發性內分泌瘤、多發性骨髓瘤、骨髓增生異常綜合征、成神經細胞瘤、神經內分泌腫瘤、卵巢癌、胰腺癌、甲狀腺乳頭狀癌、甲狀旁腺腫瘤、兒科癌症、周圍神經鞘瘤、嗜鉻細胞瘤、垂體瘤、***癌、眼色素層後黑素瘤(posterious uveal melanoma)、罕見血液學病症、腎轉移性癌症、橫紋肌樣瘤、橫紋肌肉瘤、肉瘤、皮膚癌、軟組織肉瘤、鱗狀細胞癌、胃癌、滑膜肉瘤、睾丸癌、胸腺癌、胸腺瘤、甲狀腺轉移性癌和子宮癌。The invention further relates to the use of any of the above ROR1-binding molecules in the treatment of a disease or condition associated with the expression of ROR1 or characterized by the expression of ROR1, or the treatment of a disease characterized by the expression of ROR1 by any of the above ROR1-binding molecules Use in a method of a condition, in particular, wherein the disease or condition associated with or expressed by the expression of ROR1 is cancer, more specifically wherein the cancer is selected from the group consisting of: adrenal tumors, AIDS-related cancers, Soft tissue acinar sarcoma, astrocytoma, adrenal cancer, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, B cell carcinoma, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma , chromophobe renal cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, benign fibrous histiocytoma of the skin, connective tissue hyperplasia, round cell tumor, ependymoma, Ewing's tumor, extramedullary mucinous cartilage Sarcoma, incomplete bone fiber formation, bone dysplasia, gallbladder or cholangiocarcinoma, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, liver Cell carcinoma, islet cell tumor, Kaposi's sarcoma, kidney cancer, leukemia, liposarcoma/malignant lipoma, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine tumor , multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid carcinoma, parathyroid tumor, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma , pituitary tumor, prostate cancer, posterior melanoma (posterious uveal melanoma), rare hematological disease, renal metastatic cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, Gastric cancer, synovial sarcoma, testicular cancer, thymic cancer, thymoma, metastatic thyroid cancer, and uterine cancer.

本發明涉及優化的ROR1-結合分子，其相對於現有的ROR1-結合分子具有增強的親和力和介導對腫瘤細胞的重定向細胞毒性的更好的能力。更具體地，本發明涉及包括可變輕鏈和/或可變重鏈(VH)結構域的優化的ROR1-結合分子，所述可變輕鏈和/或可變重鏈(VH)結構域已經被優化以結合存在於人ROR1多肽上的表位元，以便顯示出對人ROR1增強的結合親和力和/或在施用於接受受試者後降低的免疫原性。本發明特別涉及雙特異性，三特異性或多特異性ROR1-結合分子，包括雙特異性雙抗體、BiTEs、雙特異性抗體、三價結合分子等，其包括：(i)這樣的優化的結合ROR1的可變結構域和(ii)能夠結合存在於效應細胞表面上的分子的表位元的結構域。本發明還涉及含有任何這樣的ROR1-結合分子的藥物組合物，以及涉及包括在癌症和其它疾病和狀況的治療中使用任何這樣的ROR1-結合分子的方法。The present invention relates to an optimized ROR1-binding molecule that has enhanced affinity relative to existing ROR1-binding molecules and a better ability to mediate redirected cytotoxicity to tumor cells. More specifically, the invention relates to an optimized ROR1-binding molecule comprising a variable light chain and/or a variable heavy chain (VH) domain, said variable light chain and/or variable heavy chain (VH) domain It has been optimized to bind to epitopes present on human ROR1 polypeptides in order to exhibit enhanced binding affinity for human ROR1 and/or reduced immunogenicity upon administration to a subject. The invention particularly relates to bispecific, trispecific or multispecific ROR1-binding molecules, including bispecific diabodies, BiTEs, bispecific antibodies, trivalent binding molecules, etc., which comprise: (i) such optimized Binding to the variable domain of ROR1 and (ii) the domain of an epitope capable of binding to a molecule present on the surface of an effector cell. The invention also relates to pharmaceutical compositions containing any such ROR1-binding molecules, as well as to methods of using any such ROR1-binding molecules in the treatment of cancer and other diseases and conditions.

II .. 抗體及其結合結構域Antibody and its binding domain

本發明所述的抗體是能夠通過位於免疫球蛋白分子的可變結構域中的至少一個抗原識別位點，特異性結合靶標例如碳水化合物、多核苷酸、脂質、多肽等的免疫球蛋白分子。如本文所用的術語“抗體”(“antibody ”和“antibodies ”)指單克隆抗體、多特異性抗體、人抗體、人源化抗體、合成抗體、嵌合抗體、多克隆抗體、駱駝源化(camelized)、單鏈Fvs(scFv)、單鏈抗體、Fab片段、F(ab')片段、二硫鍵連接的雙特異性Fvs(sdFv)、胞內抗體和任何上述抗體的表位-結合片段。尤其地，術語“抗體”包括免疫球蛋白分子和免疫球蛋白分子的免疫活性片段，即含有表位-結合位點的分子。免疫球蛋白分子可以是任何類型(例如IgG、IgE、IgM、IgD、IgA和IgY)、類別(例如IgG₁ 、IgG₂ 、IgG₃ 、IgG₄ 、IgA₁ 和IgA₂ )或亞類。抗體能夠免疫特異性結合多肽或蛋白質或非蛋白質分子，因為在這樣的分子上存在特定的結構域或部分或構象(“表位 ”)。包含表位的分子可具有免疫原性活性，以便其在動物中引起抗體產生反應；這類分子稱為“抗原 ”。最近數十年已經看到對抗體的治療潛能的興趣的復興，並且抗體已經成為一種主要類型的生物技術衍生的藥物(Chan, C.E.等(2009) “The Use Of Antibodies In The Treatment Of Infectious Diseases ,” Singapore Med. J. 50(7):663-666)。超過200種基於抗體的藥物已經被批准使用或正在開發。The antibody of the present invention is an immunoglobulin molecule capable of specifically binding to a target such as a carbohydrate, a polynucleotide, a lipid, a polypeptide or the like through at least one antigen recognition site located in a variable domain of an immunoglobulin molecule. As used herein, the term "antibody" ( "Antibody" and "Antibodies") refers to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, polyclonal antibodies, camelized ( Camelized), single-chain Fvs (scFv), single-chain antibody, Fab fragment, F(ab') fragment, disulfide-linked bispecific Fvs (sdFv), intrabody, and epitope-binding fragment of any of the above antibodies . In particular, the term "antibody" includes immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, ie, molecules that contain an epitope-binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., _{_{_{IgG 1, IgG 2, IgG 3}}} , IgG 4, IgA 1 and IgA ₂₎ or subclass. An antibody is capable of immunospecifically binding to a polypeptide or protein or non-protein molecule because of the presence of a particular domain or portion or conformation (" epitope ") on such a molecule. A molecule comprising an epitope may have immunogenic activity such that it causes an antibody to produce a response in an animal; such a molecule is referred to as an " antigen ." The revival of interest in the therapeutic potential of antibodies has been seen in recent decades, and antibodies have become a major type of biotechnologically derived drug (Chan, CE et al. (2009) " The Use Of Antibodies In The Treatment Of Infectious Diseases , Singapore Med. J. 50(7): 663-666). More than 200 antibody-based drugs have been approved or are being developed.

術語“單克隆抗體”(“monoclonal antibody ”) 指同質型(homogeneous)抗體群體，其中單克隆抗體包括參與抗原的選擇性結合的氨基酸(天然產生的或非天然產生的)。單克隆抗體是高度特異性的，直接針對單個表位(或抗原位點)。術語“單克隆抗體”不僅僅包括完整的單克隆抗體和全長單克隆抗體，而且也包括其片段(比如Fab、Fab'、F(ab')₂ Fv)、單鏈(scFv)、其突變體、包括抗體部分的融合蛋白、人源化的單克隆抗體、嵌合單克隆抗體和包括具有結合抗原的必要的特異性和能力的抗原識別位點的免疫球蛋白分子的任何其他修飾構造(configuration)。就抗體的來源或製備其的方式(例如，通過雜交瘤、噬菌體選擇、重組表達、轉基因動物等)而言，不旨在是限制性的。術語包括完整的免疫球蛋白以及上面根據“抗體”的定義描述的片段等。製備單克隆抗體的方法是本領域已知的。可採用的一種方法是Kohler, G.等(1975) “Continuous Cultures Of Fused Cells Secreting Antibody Of Predefined Specificity ,” Nature 256:495-497的方法或其改良。典型地，單克隆抗體在小鼠、大鼠或兔子中開發。通過用免疫原性量的細胞、細胞提取物或包含期望的表位的蛋白質製品免疫動物而產生抗體。免疫原可以是但不限於原代細胞、培養細胞系、癌細胞、蛋白質、肽、核酸或組織。用於免疫的細胞可被培養一段時間(例如，至少24小時)，然後將它們用作免疫原。細胞它們本身或聯合非變性佐劑，比如Ribi，可用作免疫原(見，例如，Jennings, V.M. (1995) “Review of Selected Adjuvants Used in Antibody Production ,” ILAR J. 37(3):119-125)。一般而言，在用作免疫原時，細胞應保持完整並且優選地有活性。相比於破裂的細胞，完整的細胞可允許抗原被免疫的動物更好地檢測。變性或烈性佐劑，例如，弗氏佐劑的使用，可使細胞破裂，因此，其應用受阻。免疫原可以週期性間隔被多次施用，諸如兩週一次或一週一次，或者可以以維持在動物(例如，在組織重組體中)中的生存力這樣的方式被施用。可選地，對於期望的致病表位是免疫特異性的現有單克隆抗體和任意其他等價抗體可被測序，並通過本領域中已知的任意手段重組產生。在一個實施方式中，對這樣的抗體測序，然後將多核苷酸序列克隆至載體用於表達或增殖。編碼感興趣的抗體的序列可在宿主細胞中保持在載體中，並且，可然後擴展和冷凍宿主細胞，用於將來的使用。這樣的抗體的多核苷酸序列可用於基因操作，以產生本發明的單特異性或多特異性(例如，雙特異性、三特異性和四特異性)分子以及親和力優化的嵌合抗體、人源化抗體和/或犬源化(caninized)抗體，以改善抗體的親和力或其他特徵。人源化抗體的一般原理涉及保留抗體的抗原結合的部分的基本序列，同時用人抗體序列交換抗體的非人剩餘部分。The term "monoclonal antibody" ( "monoclonal antibody") refers to homogeneous (homogeneous) antibody population wherein the monoclonal antibody comprises the amino acid involved in the selective binding of an antigen (naturally occurring or non-naturally occurring). Monoclonal antibodies are highly specific and target a single epitope (or antigenic site). The term "monoclonal antibody" includes not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab') ₂ Fv), single-stranded (scFv), mutants thereof. a fusion protein comprising an antibody portion, a humanized monoclonal antibody, a chimeric monoclonal antibody, and any other modification construct comprising an immunoglobulin molecule having an antigen recognition site that binds the necessary specificity and ability of the antigen (configuration) ). The source of the antibody or the manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.) is not intended to be limiting. The term includes intact immunoglobulins as well as fragments described above in accordance with the definition of "antibody" and the like. Methods of making monoclonal antibodies are known in the art. One method that can be employed is the method of Kohler, G. et al. (1975) " Continuous Cultures Of Fused Cells Secreting Antibody Of Predefined Specificity ," Nature 256:495-497 or an improvement thereof. Typically, monoclonal antibodies are developed in mice, rats or rabbits. Antibodies are produced by immunizing an animal with an immunogenic amount of cells, cell extracts, or protein preparations containing the desired epitope. The immunogen can be, but is not limited to, a primary cell, a cultured cell line, a cancer cell, a protein, a peptide, a nucleic acid, or a tissue. The cells used for immunization can be cultured for a period of time (e.g., at least 24 hours) and then used as an immunogen. The cells themselves or in combination with non-denaturing adjuvants, such as Ribi, can be used as immunogens (see, for example, Jennings, VM (1995) " Review of Selected Adjuvants Used in Antibody Production ," ILAR J. 37(3): 119- 125). In general, when used as an immunogen, the cells should remain intact and preferably active. Intact cells can allow the antigen to be better detected by the immunized animal than the ruptured cells. Denatured or potent adjuvants, for example, the use of Freund's adjuvant, can rupture cells and, therefore, their use is hindered. The immunogen can be administered multiple times at periodic intervals, such as once every two weeks or once a week, or can be administered in a manner that maintains viability in the animal (e.g., in tissue recombinants). Alternatively, existing monoclonal antibodies and any other equivalent antibodies that are immunospecific for the desired pathogenic epitope can be sequenced and recombinantly produced by any means known in the art. In one embodiment, such an antibody is sequenced and the polynucleotide sequence is then cloned into a vector for expression or proliferation. The sequence encoding the antibody of interest can be maintained in the vector in the host cell, and the host cell can then be expanded and frozen for future use. Polynucleotide sequences of such antibodies can be used in gene manipulation to produce monospecific or multispecific (e.g., bispecific, trispecific, and tetraspecific) molecules of the invention as well as affinity optimized chimeric antibodies, humans The antibody and/or caninized antibody is conjugated to improve the affinity or other characteristics of the antibody. The general principle of a humanized antibody involves retaining the basic sequence of the antigen-binding portion of the antibody while exchanging the non-human remainder of the antibody with the human antibody sequence.

天然抗體(例如IgG抗體)是由兩條“輕鏈”和兩條“重鏈”複合而構成的。每條輕鏈包括可變結構域(“VL” )和恒定結構域(“CL” )。每條重鏈包括可變結構域(“VH” )、三個恒定結構域(“CH1” 、 “CH2” 和 “CH3” )和位於CH1 和CH2 結構域之間的“鉸鏈 ”區域(“H” )。天然產生的免疫球蛋白(例如，IgG)的基本結構單元因此是具有兩條輕鏈和兩條重鏈的四聚體，通常作為約150,000 Da的糖蛋白被表達。每條鏈的氨基端(“N- 末端 ”)部分包括主要負責抗原識別的約100至110或更多氨基酸的可變結構域。每條鏈的羧基端(“C- 末端 ”)部分限定了恒定區，其中輕鏈具有單個恒定結構域而重鏈通常具有三個恒定結構域和鉸鏈區。因此，IgG分子的輕鏈的結構是n-VL-CL-c並且IgG重鏈的結構是n-VH-CH1-H-CH2-CH3-c (其中n和c分別表示多肽的N-末端和C-末端)。IgG分子的可變結構域由互補決定區(“CDR ”)和稱為框架區段(“FR ”)的非CDR區段組成，所述互補決定區(CDR)包含與表位接觸的殘基，所述框架區段一般維持結構和決定CDR環的定位，以便允許這類接觸(儘管某些框架殘基也可接觸抗原)。因此，VL和VH結構域具有結構n-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-c 。是(或可用作)抗體的輕鏈的第一、第二和第三CDR的多肽在本文分別命名為CDR_L 1 結構域、 CDR_L 2 結構域和 CDR_L 3 結構域 。類似地，是(或可用作)抗體的重鏈的第一、第二和第三CDR的多肽在本文分別命名為CDR_H 1 結構域、 CDR _H 2 結構域和 CDR _H 3 結構域 。因此，術語CDR_L 1結構域、CDR_L 2結構域、CDR_L 3結構域、CDR_H 1結構域、CDR_H 2結構域和CDR_H 3結構域指當併入到蛋白質中時使得該蛋白質能夠結合特異性表位的多肽，無論這樣的蛋白質是否是具有輕鏈和重鏈的抗體或是雙抗體或單鏈結合分子(例如，scFv、BiTe等)或是另一類型的蛋白質。因此，如本文所使用，術語“表位結合片段 ”表示能夠免疫特異性結合表位的分子的片段。表位-結合片段可以包括抗體的任何1、2、3、4或5個CDR結構域，或者可以包括抗體的所有6個CDR結構域，並且儘管能夠免疫特異性結合這類表位，但可展示對與這樣的抗體的表位不同的這樣的表位的免疫特異性、親和力或選擇性。但是，優選地，表位結合片段將包含這類抗體的所有6個CDR結構域。抗體的表位結合片段可以是單個多肽鏈(例如，scFv)，或可包括兩條或更多條多肽鏈，每條鏈均具有氨基末端和羧基末端(例如，雙抗體、Fab片段、Fab₂ 片段等)。除非具體指出，否則本文所述的蛋白質分子的結構域的順序是從“N-末端至C-末端”方向。Natural antibodies (eg, IgG antibodies) are composed of two "light chains" and two "heavy chains". Each light chain includes a variable domain ( "VL" ) and a constant domain ( "CL" ). Each heavy chain includes a variable domain ( "VH" ), three constant domains ( "CH1" , "CH2", and "CH3" ) and a " hinge " region between the CH1 and CH2 domains ( "H " ). The basic building block of a naturally occurring immunoglobulin (eg, IgG) is thus a tetramer with two light chains and two heavy chains, typically expressed as a glycoprotein of about 150,000 Da. The amino terminus (" N- terminal ") portion of each chain includes a variable domain of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy terminus (" C- terminal ") portion of each chain defines a constant region where the light chain has a single constant domain and the heavy chain typically has three constant domains and a hinge region. Thus, the structure of the light chain of an IgG molecule is n-VL-CL-c and the structure of the IgG heavy chain is n-VH-CH1-H-CH2-CH3-c (where n and c represent the N-terminus of the polypeptide, respectively C-end). The variable domain of an IgG molecule consists of a complementarity determining region (" CDR ") and a non-CDR segment called a framework segment (" FR "), which contains residues that are in contact with the epitope. The framework segments generally maintain structure and determine the location of the CDR loops to allow for such contacts (although certain framework residues can also contact the antigen). Thus, the VL and VH domains have the structure n-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-c . Polypeptides that are (or can be used as) the first, second, and third CDRs of the light chain of an antibody are designated herein as CDR _L 1 domain, CDR _L 2 domain, and CDR _L 3 domain, respectively . Similarly, polypeptides that are (or can be used as) the first, second, and third CDRs of the heavy chain of an antibody are designated herein as CDR _H 1 domain, CDR _H 2 domain, and CDR _H 3 domain, respectively . Thus, the terms CDR _L 1 domain, CDR _L 2 domain, CDR _L 3 domain, CDR _H 1 domain, CDR _H 2 domain and CDR _H 3 domain mean that the protein is capable of being incorporated into a protein A polypeptide that binds to a specific epitope, whether or not such a protein is an antibody having a light chain and a heavy chain or a diabody or a single-stranded binding molecule (eg, scFv, BiTe, etc.) or another type of protein. Thus, as used herein, the term " epitope binding fragment " refers to a fragment of a molecule that is capable of immunospecifically binding an epitope. An epitope-binding fragment can comprise any 1, 2, 3, 4 or 5 CDR domains of an antibody, or can comprise all 6 CDR domains of an antibody, and although immunospecifically binds to such an epitope, The immunospecificity, affinity or selectivity of such epitopes that differ from the epitope of such antibodies is shown. Preferably, however, the epitope binding fragment will comprise all six CDR domains of such antibodies. An epitope binding fragment of an antibody can be a single polypeptide chain (eg, an scFv), or can include two or more polypeptide chains, each having an amino terminus and a carboxy terminus (eg, a diabody, a Fab fragment, a Fab ₂₎ Fragments, etc.). Unless specifically stated otherwise, the order of the domains of the protein molecules described herein is from the "N-terminal to C-terminal" orientation.

本發明尤其包括包含本發明優化的抗-ROR1-VL結構域和/或VH結構域的單鏈可變結構域片段(“ scFv ” )和包含本發明優化的抗-ROR1-VL結構域和/或VH結構域的多特異性結合分子。單鏈可變結構域片段包括利用短的“連接體”肽連接在一起的VL和VH結構域。這樣的連接體可以被修飾以提供另外的功能，例如允許藥物的附著或允許附著到固體載體。可經重組或合成產生單鏈變體。為了合成產生scFv，可使用自動合成儀。為了重組產生scFv，可將包含編碼scFv的多核苷酸的適當的質粒引入到適當的宿主細胞中，所述宿主細胞是真核細胞，比如酵母細胞、植物細胞、昆蟲細胞或哺乳動物細胞，或原核細胞，比如大腸埃希氏菌(E. coli )。可通過常規操作比如多核苷酸的連接製備編碼感興趣的scFv的多核苷酸。可使用本領域已知的標準蛋白質純化技術分離所得scFv。The invention particularly includes single-chain variable domain fragments ( " scFv " ) comprising an optimized anti-ROR1-VL domain and/or VH domain of the invention and an optimized anti-ROR1-VL domain comprising the invention and/or Or a multispecific binding molecule of the VH domain. Single-chain variable domain fragments include VL and VH domains joined together using short "linker" peptides. Such linkers can be modified to provide additional functionality, such as allowing attachment of the drug or allowing attachment to a solid carrier. Single-chain variants can be produced recombinantly or synthetically. For the synthesis to produce scFv, an automated synthesizer can be used. For recombinant production of scFv, a suitable plasmid comprising a polynucleotide encoding an scFv can be introduced into a suitable host cell, such as a yeast cell, a plant cell, an insect cell or a mammalian cell, or Prokaryotic cells, such as E. coli . Polynucleotides encoding the scFv of interest can be prepared by conventional manipulations such as ligation of polynucleotides. The resulting scFv can be isolated using standard protein purification techniques known in the art.

本發明還尤其包括包含人源化抗體的抗-ROR1-VL結構域和/或VH結構域的優化的ROR1-結合分子。術語“人源化 ” 抗體指一般使用重組技術製備的嵌合分子，其具有來自非人物種的免疫球蛋白的表位結合位點和基於人免疫球蛋白的結構和/或序列的分子的剩餘免疫球蛋白結構。這類抗體的可變結構域的多核苷酸序列可用於遺傳操作，以產生這類衍生物和改善這類抗體的親和力或其他特徵。人源化抗體的一般原則涉及保留抗體的表位元結合部分的基本序列，同時用人抗體序列交換抗體的非人剩餘部分。人源化單克隆抗體有四個基本步驟。這些是：(1)確定開始抗體輕鏈和重鏈可變結構域的核苷酸和預測的氨基酸序列；(2)設計人源化抗體或犬源化抗體，即，決定在人源化或犬源化過程中使用哪種抗體框架區域；(3)實際人源化或犬源化方法/技術；和(4)轉染和表達人源化抗體。見，例如，美國專利號4,816,567、5,807,715、5,866,692和6,331,415。The invention also includes, inter alia, an optimized ROR1-binding molecule comprising an anti-ROR1-VL domain and/or a VH domain of a humanized antibody. The term " humanized " antibody refers to a chimeric molecule, typically prepared using recombinant techniques, having an epitope binding site for an immunoglobulin from a non-human species and a remaining molecule based on the structure and/or sequence of the human immunoglobulin. Immunoglobulin structure. The polynucleotide sequences of the variable domains of such antibodies can be used in genetic manipulation to produce such derivatives and to improve the affinity or other characteristics of such antibodies. The general principle of humanized antibodies involves retaining the basic sequence of the epitope binding portion of the antibody while exchanging the non-human remainder of the antibody with the human antibody sequence. Humanized monoclonal antibodies have four basic steps. These are: (1) determining the nucleotide and predicted amino acid sequence of the start of the antibody light and heavy chain variable domains; (2) designing a humanized antibody or canineized antibody, ie, deciding in humanization or Which antibody framework regions are used during canineization; (3) actual humanization or canineization methods/techniques; and (4) transfection and expression of humanized antibodies. See, for example, U.S. Patent Nos. 4,816,567, 5,807,715, 5,866,692, and 6,331,415.

表位-結合位點可包括融合到恒定結構域融合上的完整的可變結構域或僅僅包括移植至適當框架區的這類可變結構域的互補決定區(CDR)。表位結合位點可以是野生型或通過一個或多個氨基酸取代被修飾。這消除了人個體中作為免疫原的恒定區，但是仍保留對外源可變結構域的免疫應答的可能性(LoBuglio, A.F.等(1989) “Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response ,” Proc. Natl. Acad. Sci. (U.S.A.) 86:4220-4224)。另一種方法不僅僅關注提供源自人的恒定區，而且也修飾可變結構域，以便重塑它們使其盡可能地與人形式接近。已知重鏈和輕鏈的可變結構域都包含三個互補決定區(CDR)，側翼是四個框架區(FR)，所述互補決定區(CDR)對所討論的抗原回應不同並且決定結合能力，所述框架區(FR)在給定物種中相對保守並且推定其為CDR提供支架。當針對特定抗原製備非人抗體時，通過將源自非人抗體的CDR移植在待修飾的人抗體中存在的FR上可“重塑”或“人源化”可變結構域。已經報導了該方法應用於各種抗體：Sato, K.等(1993) Cancer Res 53:851-856. Riechmann, L.等(1988) “Reshaping Human Antibodies for Therapy ,” Nature 332:323-327；Verhoeyen, M.等(1988) “Reshaping Human Antibodies: Grafting An Antilysozyme Activity ,” Science 239:1534-1536；Kettleborough, C. A.等(1991) “Humanization Of A Mouse Monoclonal Antibody By CDR-Grafting: The Importance Of Framework Residues On Loop Conformation ,” Protein Engineering 4:773-3783；Maeda, H.等(1991) “Construction Of Reshaped Human Antibodies With HIV-Neutralizing Activity ,” Human Antibodies Hybridoma 2:124-134；Gorman, S. D.等(1991) “Reshaping A Therapeutic CD4 Antibody ,” Proc. Natl. Acad. Sci. (U.S.A.) 88:4181-4185；Tempest, P.R.等(1991) “Reshaping A Human Monoclonal Antibody To Inhibit Human Respiratory Syncytial Virus Infection in vivo ,” Bio/Technology 9:266-271；Co, M. S.等(1991) “Humanized Antibodies For Antiviral Therapy ,” Proc. Natl. Acad. Sci. (U.S.A.) 88:2869-2873；Carter, P.等(1992) “Humanization Of An Anti-p185her2 Antibody For Human Cancer Therapy ,” Proc. Natl. Acad. Sci. (U.S.A.) 89:4285-4289；和Co, M.S.等(1992) “Chimeric And Humanized Antibodies With Specificity For The CD33 Antigen ,” J. Immunol. 148:1149-1154。在一些實施方式中，人源化抗體保留所有的CDR序列(例如，人源化鼠抗體，其包含來自小鼠抗體的所有六個CDR)。在其他實施方式中，人源化抗體具有一個或多個CDR (一個、兩個、三個、四個、五個或六個)，其序列相對於初始抗體不同。An epitope-binding site can include a complete variable domain fused to a constant domain fusion or only a complementarity determining region (CDR) of such a variable domain that is grafted to the appropriate framework region. The epitope binding site can be wild type or modified by one or more amino acid substitutions. This eliminates the constant region of the human individual as an immunogen, but still retains the possibility of an immune response to the foreign variable domain (LoBuglio, AF et al. (1989) " Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response ," Proc. Natl. Acad. Sci. (USA) 86:4220-4224). Another approach is not only concerned with providing constant regions derived from humans, but also modifying the variable domains in order to reshape them to be as close as possible to human forms. It is known that the variable domains of both heavy and light chains comprise three complementarity determining regions (CDRs) flanked by four framework regions (FRs) that respond differently to the antigen in question and determine The binding capacity, the framework region (FR) is relatively conserved in a given species and is presumed to provide a scaffold for the CDRs. When a non-human antibody is prepared for a particular antigen, the variable domain can be "reshaped" or "humanized" by grafting the CDR derived from the non-human antibody onto the FR present in the human antibody to be modified. This method has been reported for use in various antibodies: Sato, K. et al. (1993) Cancer Res 53: 851-856. Riechmann, L. et al. (1988) " Reshaping Human Antibodies for Therapy ," Nature 332: 323-327; Verhoeyen , M. et al. (1988) “ Reshaping Human Antibodies: Grafting An Antilysozyme Activity ,” Science 239: 1534-1536; Kettleborough, CA et al. (1991) “ Humanization Of A Mouse Monoclonal Antibody By CDR-Grafting: The Importance Of Framework Residues On Loop Conformation ," Protein Engineering 4: 773-3783; Maeda, H. et al. (1991) " Configuration Of Reshaped Human Antibodies With HIV-Neutralizing Activity ," Human Antibodies Hybridoma 2: 124-134; Gorman, SD et al. (1991) Reshaping A Therapeutic CD4 Antibody ," Proc. Natl. Acad. Sci. (USA) 88:4181-4185; Tempest, PR et al. (1991) " Reshaping A Human Monoclonal Antibody To Inhibit Human Respiratory Syncytial Virus Infection in vivo ," Bio/ Technology 9: 266-271; Co, MS et al. (1991) " Humanized Antibodies For Antiviral Therapy ," Proc. Natl. Acad. Sci. (USA) 88:2869- 2873; Carter, P. et al. (1992) " Humanization Of An Anti-p185her2 Antibody For Human Cancer Therapy ," Proc. Natl. Acad. Sci. (USA) 89: 4285-4289; and Co, MS et al. (1992) Chimeric And Humanized Antibodies With Specificity For The CD33 Antigen ," J. Immunol. 148: 1149-1154. In some embodiments, the humanized antibody retains all of the CDR sequences (eg, a humanized murine antibody comprising all six CDRs from a mouse antibody). In other embodiments, the humanized antibody has one or more CDRs (one, two, three, four, five or six), the sequence of which is different from the original antibody.

已經描述了包括源自非人免疫球蛋白的表位結合位點的許多人源化抗體分子，包括具有齧齒動物或修飾的齧齒動物可變結構域和它們與人恒定結構域融合的相關的互補決定區(CDR)的嵌合抗體(見，例如，Winter等(1991) “Man-made Antibodies ,” Nature 349:293-299；Lobuglio等(1989) “Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response ,” Proc. Natl. Acad. Sci. (U.S.A.) 86:4220-4224 (1989), Shaw等(1987) “Characterization Of A Mouse/Human Chimeric Monoclonal Antibody (17-1A) To A Colon Cancer Tumor-Associated Antigen ,” J. Immunol. 138:4534-4538, and Brown等(1987) “Tumor-Specific Genetically Engineered Murine/Human Chimeric Monoclonal Antibody ,” Cancer Res. 47:3577-3583)。其他參考文獻描述了移植至人支撐框架區(FR)的齧齒動物CDR，其然後與適當的人抗體恒定結構域融合(見，例如，Riechmann, L.等(1988) “Reshaping Human Antibodies for Therapy ,” Nature 332:323-327；Verhoeyen, M.等(1988) “Reshaping Human Antibodies: Grafting An Antilysozyme Activity ,” Science 239:1534-1536；和Jones等(1986) “Replacing The Complementarity-Determining Regions In A Human Antibody With Those From A Mouse ,” Nature 321:522-525)。另外的參考文獻描述了由重組修飾的齧齒動物框架區支撐的齧齒動物CDR。見，例如，歐洲專利公開號519,596。這些“人源化的”分子被設計，以使對齧齒動物抗人抗體分子的不利免疫應答最小化，所述不利免疫應答限制了這些部分在人接受者中治療應用的持續時間和效力。也可使用的人源化抗體的其他方法，其公開在以下文獻中：Daugherty等(1991) “Polymerase Chain Reaction Facilitates The Cloning, CDR-Grafting, And Rapid Expression Of A Murine Monoclonal Antibody Directed Against The CD18 Component Of Leukocyte Integrins ,” Nucl. Acids Res. 19:2471-2476和美國專利號6,180,377、6,054,297、5,997,867和5,866,692。A number of humanized antibody molecules comprising epitope binding sites derived from non-human immunoglobulins have been described, including rodent variable domains with rodent or modification and their associated complementation to human constant domain fusion Chimeric antibodies that determine the region (CDR) (see, for example, Winter et al. (1991) " Man-made Antibodies ," Nature 349:293-299; Lobuglio et al. (1989) " Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response ," Proc. Natl. Acad. Sci. (USA) 86:4220-4224 (1989), Shaw et al. (1987) "Functionization Of A Mouse/Human Chimeric Monoclonal Antibody (17-1A) To A Colon Cancer Tumor- Associated Antigen ," J. Immunol. 138:4534-4538, and Brown et al. (1987) " Tumor-Specific Genetically Engineered Murine/Human Chimeric Monoclonal Antibody ," Cancer Res. 47:3577-3583). Other references describe rodent CDRs grafted to the human support framework region (FR), which are then fused to appropriate human antibody constant domains (see, for example, Riechmann, L. et al. (1988) " Reshaping Human Antibodies for Therapy , Nature 332: 323-327; Verhoeyen, M. et al. (1988) “ Reshaping Human Antibodies: Grafting An Antilysozyme Activity ,” Science 239: 1534-1536; and Jones et al. (1986) “ Replacing The Complementarity-Determining Regions In A Human Antibody With Those From A Mouse ," Nature 321:522-525). Additional references describe rodent CDRs supported by recombinantly modified rodent framework regions. See, for example, European Patent Publication No. 519,596. These "humanized" molecules are designed to minimize adverse immune responses to rodent anti-human antibody molecules that limit the duration and efficacy of therapeutic applications of these moieties in human recipients. Other methods of humanized antibodies that can also be used are disclosed in Daugherty et al. (1991) " Polymerase Chain Reaction Facilitates The Cloning, CDR-Grafting, And Rapid Expression Of A Murine Monoclonal Antibody Directed Against The CD18 Component Of Leukocyte Integrins , "Nucl. Acids Res. 19: 2471-2476 and U.S. Patent Nos. 6,180,377, 6,054,297, 5,997,867 and 5,866,692.

II .Fcγ 受體 (FcγRs) II. Fcγ receptors (FcyRs)

兩條重鏈的CH2和CH3結構域相互作用形成“Fc 區域 ”，其是被細胞Fc受體(包括但不限於Fcγ受體(FcγRs))識別的結構域。如本文所用，術語“Fc區域”用於定義IgG重鏈的C-末端區域。如果Fc區域的氨基酸序列相對於其它IgG同種型與特定的同種型最同源，則認為該Fc區域屬於該IgG同種型、類或亞類。除了它們在診斷中的已知用途，已經顯示抗體可用作治療劑。The CH2 and CH3 domains of the two heavy chains interact to form an " Fc region " which is a domain recognized by cellular Fc receptors including, but not limited to, Fc gamma receptors (FcyRs). As used herein, the term "Fc region" is used to define the C-terminal region of an IgG heavy chain. An Fc region is considered to belong to the IgG isotype, class or subclass if the amino acid sequence of the Fc region is most homologous to the other IgG isoform and the particular isoform. In addition to their known use in diagnosis, antibodies have been shown to be useful as therapeutic agents.

示例性的人IgG1的CH2-CH3結構域的氨基酸序列是(SEQ ID NO:1 )： 231 240 250 260 270 280 APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD 290 300 310 320 330 GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA 340 350 360 370 380 PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE 390 400 410 420 430 WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE 440 447 ALHNHYTQKS LSLSPG X 如Kabat中闡釋的EU索引編號，其中X是賴氨酸(K)或不存在。The amino acid sequence of the CH2-CH3 domain of an exemplary human IgG1 is ( SEQ ID NO: 1 ): 231 240 250 260 270 280 APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD 290 300 310 320 330 GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA 340 350 360 370 380 PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE 390 400 410 420 430 WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE 440 447 ALHNHYTQKS LSLSPG X EU index number as explained in Kabat, where X is lysine (K) or absent.

示例性的人IgG2的CH2-CH3結構域的氨基酸序列是(SEQ ID NO:2) ： 231 240 250 260 270 280 APPVA-GPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFNWYVD 290 300 310 320 330 GVEVHNAKTK PREEQFNSTF RVVSVLTVVH QDWLNGKEYK CKVSNKGLPA 340 350 360 370 380 PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDISVE 390 400 410 420 430 WESNGQPENN YKTTPPMLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE 440 447 ALHNHYTQKS LSLSPG X 如Kabat中闡釋的EU索引編號，其中X是賴氨酸(K)或不存在。The amino acid sequence of the CH2-CH3 domain of an exemplary human IgG2 is (SEQ ID NO: 2) : 231 240 250 260 270 280 APPVA-GPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFNWYVD 290 300 310 GVEVHNAKTK PREEQFNSTF RVVSVLTVVH QDWLNGKEYK CKVSNKGLPA 340 350 360 370 380 PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDISVE 390 400 410 420 430 WESNGQPENN YKTTPPMLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE 440 447 ALHNHYTQKS LSLSPG X EU index number as explained in Kabat, where X is lysine (K) or absent.

示例性的人IgG3的CH2-CH3結構域的氨基酸序列是(SEQ ID NO:3) ： 231 240 250 260 270 280 APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFKWYVD 290 300 310 320 330 GVEVHNAKTK PREEQYNSTF RVVSVLTVLH QDWLNGKEYK CKVSNKALPA 340 350 360 370 380 PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE 390 400 410 420 430 WESSGQPENN YNTTPPMLDS DGSFFLYSKL TVDKSRWQQG NIFSCSVMHE 440 447 ALHNRFTQKS LSLSPG X 如Kabat中闡釋的EU索引編號，其中X是賴氨酸(K)或不存在。The amino acid sequence of the CH2-CH3 domain of an exemplary human IgG3 is (SEQ ID NO: 3) : 231 240 250 260 270 280 APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFKWYVD 290 300 310 320 330 GVEVHNAKTK PREEQYNSTF RVVSVLTVLH QDWLNGKEYK CKVSNKALPA 340 350 360 370 380 PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE 390 400 410 420 430 WESSGQPENN YNTTPPMLDS DGSFFLYSKL TVDKSRWQQG NIFSCSVMHE 440 447 ALHNRFTQKS LSLSPG X EU index number as explained in Kabat, where X is lysine (K) or absent.

示例性的人IgG4的CH2-CH3結構域的氨基酸序列是(SEQ ID NO:4) ： 231 240 250 260 270 280 APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD 290 300 310 320 330 GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS 340 350 360 370 380 SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE 390 400 410 420 430 WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE 440 447 ALHNHYTQKS LSLSLG X 如Kabat中闡釋的EU索引編號，其中X是賴氨酸(K)或不存在。The amino acid sequence of the CH2-CH3 domain of an exemplary human IgG4 is (SEQ ID NO: 4) : 231 240 250 260 270 280 APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD 290 300 310 320 330 GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS 340 350 360 370 380 SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE 390 400 410 420 430 WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE 440 447 ALHNHYTQKS LSLSLG X EU index number as explained in Kabat, where X is lysine (K) or absent.

遍及本說明書，IgG重鏈的恒定區中殘基的編號是如在Kabat等, Sequences of Proteins of Immunological Interest, 第五版. Public Health Service, NH1, MD (1991) (“Kabat”)中的EU索引的編號，其通過參考明確併入本文。術語“如在Kabat的EU索引”指人IgG1 EU抗體的恒定結構域的編號。通過鏈中氨基酸的位置命名來自免疫球蛋白的成熟重鏈和輕鏈的可變結構域的氨基酸。Kabat描述了抗體的許多氨基酸序列，鑒定了每個亞組的氨基酸共有序列，並且為每個氨基酸賦予殘基編號，並且，如Kabat定義的來鑒定CDR (應當理解，如Chothia, C. ＆Lesk, A.M. ((1987)“Canonical Structures For The Hypervariable Regions Of Immunoglobulins, ” J.Mol.Biol.196: 901-917) 限定的CDR_H 1在之前的五個殘基開始)。通過參考保守的氨基酸比對所討論的抗體與Kabat中的共有序列中的一條，Kabat的編號方案可擴展至未包括在其綱要中的抗體。用於賦予殘基編號的該方法已經成為本領域的標準並且容易鑒定在不同抗體，包括嵌合或人源化的變體中等同位置處的氨基酸。例如，在人抗體輕鏈50位的氨基酸佔據與在小鼠抗體輕鏈的50位氨基酸的等同位置。Throughout the specification, the numbering of residues in the constant region of an IgG heavy chain is EU as in Kabat et al , Sequences of Proteins of Immunological Interest, Fifth Edition. Public Health Service, NH1, MD (1991) ("Kabat"). The numbering of the index, which is expressly incorporated herein by reference. The term "EU index as in Kabat" refers to the numbering of the constant domains of human IgGl EU antibodies. The amino acids from the variable domains of the mature heavy and light chains of immunoglobulins are named by the position of the amino acids in the chain. Kabat describes a number of amino acid sequences of antibodies, identifies the amino acid consensus sequences for each subgroup, and assigns a residue number to each amino acid, and, as defined by Kabat, identifies the CDRs (should be understood, such as Chothia, C. & Lesk, AM ((1987) " Canonical Structures For The Hypervariable Regions Of Immunoglobulins, " J. Mol. Biol. 196: 901-917) The defined CDR _H 1 begins with the first five residues). By reference to a conserved amino acid alignment of one of the consensus antibodies in the consensus sequence in Kabat, Kabat's numbering scheme can be extended to antibodies not included in its profile. This method for assigning residue numbers has become a standard in the art and readily identifies amino acids at equivalent positions in different antibodies, including chimeric or humanized variants. For example, an amino acid at position 50 of the human antibody light chain occupies an equivalent position to the 50 amino acid of the mouse antibody light chain.

已經在抗體恒定區中的許多不同位置(例如，Fc位置，包括但不限於270、272、312、315、356和358位，如在Kabat闡釋的EU索引編號)處觀察到了多態性，因此示出的序列和現有技術的序列之間可能存在輕微的差別。已經充分表徵了人免疫球蛋白的多態性形式。目前，已知18個Gm同種異型：G1m (1、2、3、17)或G1m (a、x、f、z)、G2m (23)或G2m (n)、G3m (5、6、10、11、13、14、15、16、21、24、26、27、28)或G3m (b1、c3、b3、b0、b3、b4、s、t、g1、c5、u、v、g5) (Lefranc, 等, “The Human IgG Subclasses: Molecular Analysis Of Structure, Function And Regulation. ”Pergamon, Oxford, pp. 43-78 (1990)；Lefranc, G. 等1979，Hum. Genet.: 50, 199-211)。具體考慮本發明的抗體可併入任何免疫球蛋白基因的任何同種異型(allotype)、異同種異型(isoallotype)或單倍型(haplotype)，並且不限於本文提供的序列的同種異型、異同種異型或單倍型。此外，在一些表達體系中，CH3結構域的C-末端氨基酸殘基(上面粗體顯示的)可在翻譯後去除。因此，CH3結構域的C-末端殘基是本發明的ROR1-結合分子中的任選的氨基酸殘基。本發明尤其包括缺乏CH3結構域的C-末端殘基的ROR1-結合分子。本發明還尤其包括的是包括CH3結構域的C-末端賴氨酸殘基的這類構建體。Polymorphisms have been observed at many different positions in the constant region of the antibody (eg, Fc positions, including but not limited to positions 270, 272, 312, 315, 356, and 358, as in the EU index number interpreted by Kabat) There may be a slight difference between the sequence shown and the prior art sequence. Polymorphic forms of human immunoglobulin have been well characterized. At present, 18 Gm allotypes are known: G1m (1, 2, 3, 17) or G1m (a, x, f, z), G2m (23) or G2m (n), G3m (5, 6, 10, 11, 13, 14, 15, 16, 21, 24, 26, 27, 28) or G3m (b1, c3, b3, b0, b3, b4, s, t, g1, c5, u, v, g5) Lefranc, et al, " The Human IgG Subclasses: Molecular Analysis Of Structure, Function And Regulation. " Pergamon, Oxford, pp. 43-78 (1990); Lefranc, G. et al. 1979, Hum. Genet.: 50, 199-211 ). It is specifically contemplated that an antibody of the invention may be incorporated into any allotype, isoallotype, or haplotype of any immunoglobulin gene, and is not limited to allotypes, isoforms, and isoforms of the sequences provided herein. Or haplotype. Furthermore, in some expression systems, the C-terminal amino acid residues of the CH3 domain (shown in bold above) can be removed post-translationally. Thus, the C-terminal residue of the CH3 domain is an optional amino acid residue in the ROR1-binding molecule of the invention. The invention particularly includes ROR1-binding molecules that lack the C-terminal residue of the CH3 domain. Also included within the invention are such constructs comprising a C-terminal lysine residue of the CH3 domain.

如上所述，天然IgG抗體的Fc區能夠結合細胞Fc γ受體( FcγR) 。這類結合導致啟動或抑制信號向免疫系統的傳導。導致截然相反的功能的這類結合的能力反映了不同FcγR之間的結構差異，尤其反映了結合的FcγR是否具有基於免疫受體酪氨酸的啟動基序(“ITAM”)或基於免疫受體酪氨酸的抑制基序(“ITIM”)。不同細胞質酶向這些結構的募集控制了FcγR-介導的細胞應答的結果。包含ITAM的FcγRs包括FcγRI、FcγRIIA、FcγRIIIA，並且，當結合Fc區(例如，存在於免疫複合物中的聚集Fc區域)時啟動免疫系統。FcγRIIB是目前已知的唯一包含ITIM的天然FcγR；當結合至聚集的Fc區時，其用於阻礙或抑制免疫系統。人嗜中性粒細胞表達FcγRIIA基因。通過免疫複合物或特異性抗體交聯的FcγRIIA聚類用於使ITAM與促進ITAM磷酸化的受體相關的激酶聚集。ITAM磷酸化充當Syk激酶的停泊位點，Syk激酶的啟動導致下游底物(例如，PI₃ K)的啟動。細胞啟動導致促炎性介質的釋放。FcγRIIB基因在B淋巴細胞上表達；其細胞外結構域與FcγRIIA是96%一致的，並且以不能區分的方式結合IgG複合物。ITIM在FcγRIIB的胞質結構域中的存在限定了FcγR的這種抑制性亞類。最近，確定了這種抑制的分子基礎。當與啟動FcγR共連接時，FcγRIIB中的ITIM變成磷酸化的，並且吸引肌醇聚磷酸鹽5’-磷酸酶(SHIP)的SH2結構域，肌醇聚磷酸鹽5’-磷酸酶(SHIP)水解由於含ITAM的FcγR介導的酪氨酸激酶啟動而釋放的磷酸肌醇信使，從而防止細胞內Ca++的流入。因此，FcγRIIB的交聯抑制對FcγR連接的啟動應答並抑制細胞應答。B-細胞啟動、B-細胞增殖和抗體分泌因此被中止。As described above, the Fc region of a native IgG antibody is capable of binding to the cellular Fc gamma receptor ( FcγR ) . This type of binding results in the initiation or inhibition of the transmission of signals to the immune system. The ability to cause such binding of diametrically opposite functions reflects structural differences between different FcyRs, particularly reflecting whether the bound FcyR has an immunoreceptor tyrosine-based initiation motif ("ITAM") or an immunoreceptor. Tyrosine inhibition motif ("ITIM"). The recruitment of these cytoplasmic enzymes to these structures controls the results of FcγR-mediated cellular responses. FcγRs comprising ITAM include FcγRI, FcγRIIA, FcγRIIIA, and initiate the immune system when binding to an Fc region (eg, an aggregated Fc region present in an immune complex). Fc[gamma]RIIB is the only known native Fc[gamma]R containing ITIM; it is used to block or inhibit the immune system when bound to the aggregated Fc region. Human neutrophils express the FcyRIIA gene. FcyRIIA clustering by cross-linking of immune complexes or specific antibodies was used to aggregate ITAM with kinases that are involved in receptors that promote ITAM phosphorylation. ITAM phosphorylation acts as a docking site for Syk kinase, Syk kinase activation causes start of downstream substrates (e.g., PI ₃ K) of. Cellular initiation results in the release of pro-inflammatory mediators. The FcγRIIB gene is expressed on B lymphocytes; its extracellular domain is 96% identical to FcγRIIA, and the IgG complex is bound in an indistinguishable manner. The presence of ITIM in the cytoplasmic domain of FcyRIIB defines this inhibitory subclass of FcyR. Recently, the molecular basis of this inhibition has been determined. ITIM in FcyRIIB becomes phosphorylated when co-ligated with the FcγR, and attracts the SH2 domain of inositol polyphosphate 5'-phosphatase (SHIP), inositol polyphosphate 5'-phosphatase (SHIP) Hydrolysis of the phosphoinositide messenger released by the ITAM-containing FcγR-mediated tyrosine kinase initiation prevents the influx of intracellular Ca++. Thus, cross-linking of FcyRIIB inhibits the initiation of FcγR ligation and inhibits cellular responses. B-cell initiation, B-cell proliferation and antibody secretion are therefore discontinued.

IIIIII .. 雙特異性抗體、多特異性雙抗體和Bispecific antibodies, multispecific diabodies and DART®DART® 雙抗體Double antibody

抗體結合抗原的表位的能力取決於抗體的VL和VH結構域的存在和氨基酸序列。抗體的輕鏈和重鏈的相互作用，尤其地，其VL和VH結構域的相互作用，形成天然抗體，諸如IgG，的兩個表位結合位點之一。天然抗體能夠結合僅僅一個表位種類(即，它們是單特異性的)，但是它們可結合該種類的多個拷貝(即，展示雙價或多價)。The ability of an antibody to bind to an epitope of an antigen depends on the presence of the VL and VH domains of the antibody and the amino acid sequence. The interaction of the light and heavy chains of an antibody, in particular, the interaction of its VL and VH domains, forms one of the two epitope binding sites of a native antibody, such as IgG. Natural antibodies are capable of binding to only one epitope species (ie, they are monospecific), but they can bind multiple copies of the species (ie, exhibit bivalent or multivalent).

本發明的結合結構域以“免疫特異性 ”方式結合表位元。如本文所使用，如果抗體、雙抗體或其他表位結合分子，相對於可選的表位，與另一分子的區域(即，表位元)更經常、更快速、以更長的持久性和/或以更大的親和力反應或締合，則認為抗體、雙抗體或其他表位結合分子“免疫特異性 ”結合該表位。例如，免疫特異性結合病毒表位元的抗體是，相比其免疫特異性結合其他病毒表位元或非病毒表位元，以更大的親和力、親合力、更容易和/或以更大的持久性結合該病毒表位元的抗體。通過閱讀該定義，應理解，例如，免疫特異性結合第一靶的抗體(或部分或表位元)可特異性或非特異性或優選或非優選結合第二靶。因此，“免疫特異性結合”不必要求(儘管其可包括)排他性結合。一般而言，但不是必要的，提及結合意思是“免疫特異性”結合。如果兩個分子的結合展示受體結合它們各自配體的特異性，則認為兩個分子能夠彼此以“物理 特異性 ”方式結合。The binding domains of the invention bind to epitopes in an " immunospecific " manner. As used herein, an antibody, diabody or other epitope binding molecule, with respect to an optional epitope, is more frequent, faster, and longer lasting than a region of another molecule (ie, an epitope). and / or with greater affinity reaction or association, it is considered an antibody, diabody, or another epitope binding molecule "immunospecific" binding epitope. For example, an antibody that immunospecifically binds to a viral epitope is greater affinity, affinity, easier, and/or larger than its immunospecific binding to other viral epitopes or non-viral epitopes. Persistence of antibodies that bind to the viral epitope. By reading this definition, it is understood that, for example, an antibody (or portion or epitope) that immunospecifically binds to a first target can be specifically or non-specifically or preferably or non-preferably bound to a second target. Thus, "immune specific binding" does not necessarily require (although it may include) exclusive binding. In general, but not necessarily, reference to binding means "immunospecific" binding. If the binding of two molecules exhibits the specificity of the receptors binding to their respective ligands, then the two molecules are believed to be able to bind to each other in a " physically specific " manner.

可以通過產生可同時結合兩種分開的和不同的抗原(或相同抗原的不同表位)的基於多特異性抗體的分子和/或通過產生對於相同的表位和/或抗原的具有更高價(即大於兩個結合位點)的基於抗體的分子而增強抗體的功能性。Multivalent antibody-based molecules that can simultaneously bind two separate and distinct antigens (or different epitopes of the same antigen) and/or by generating higher prices for the same epitope and/or antigen ( That is, antibody-based molecules that are greater than two binding sites) enhance the functionality of the antibody.

為了提供比天然抗體具有更大能力的分子，已經開發了各種重組雙特異性抗體形式(見，例如，PCT公佈號WO 2008/003116、WO 2009/132876、WO 2008/003103、WO 2007/146968、WO 2009/018386、WO 2012/009544、WO 2013/070565)，其大部分使用連接肽，所述連接肽與其他表位結合片段(例如，scFv、VL、VH等)融合或在抗體核心中(IgA、IgD、IgE、IgG或IgM)或與多個表位結合片段(例如，兩個Fab片段或scFvs)融合。可選的形式使用連接肽，以將表位結合片段(例如，scFv、VL、VH等)與二聚化結構域，比如CH2-CH3結構域，或可選的多肽融合(WO 2005/070966、WO 2006/107786A WO 2006/107617A、WO 2007/046893)。PCT公開號WO 2013/174873、WO 2011/133886和WO 2010/136172公開了三特異性抗體，其中CL和CH1結構域由其各自的天然位置被轉變，並且VL和VH結構域已經被多樣化(WO 2008/027236、WO 2010/108127)，以允許它們結合大於一種抗原。PCT公開號WO 2013/163427和WO 2013/119903公開了修飾CH2結構域，以包含包括結合結構域的融合蛋白加合物。PCT公開號WO 2010/028797、WO2010028796和WO 2010/028795公開了重組抗體，其Fc區已經用另外的VL和VH結構域替換，以便形成三價結合分子。PCT公開號WO 2003/025018和WO2003012069公開了重組雙抗體，其單個鏈包含scFv結構域。PCT公開號WO 2013/006544公開了多價Fab分子，其作為單多肽鏈被合成，然後經歷蛋白酶解，以產生異源二聚化結構。PCT公開號WO 2014/022540、WO 2013/003652、WO 2012/162583、WO 2012/156430、WO 2011/086091、WO 2008/024188、WO 2007/024715、WO 2007/075270、WO 1998/002463、WO 1992/022583和WO 1991/003493公開了添加另外的結合結構域或功能集團至抗體或抗體部分(例如，添加雙抗體至抗體的輕鏈或添加另外的VL和VH結構域至抗體的輕鏈和重鏈或添加異源融合蛋白或彼此連結多個Fab結構域)。In order to provide molecules with greater capacity than natural antibodies, various recombinant bispecific antibody formats have been developed (see, for example, PCT Publication Nos. WO 2008/003116, WO 2009/132876, WO 2008/003103, WO 2007/146968, WO 2009/018386, WO 2012/009544, WO 2013/070565), the majority of which use a linker peptide fused to other epitope binding fragments (eg, scFv, VL, VH, etc.) or in an antibody core ( IgA, IgD, IgE, IgG or IgM) or fused to multiple epitope binding fragments (eg, two Fab fragments or scFvs). An alternative format uses a linker peptide to fuse an epitope binding fragment (eg, scFv, VL, VH, etc.) to a dimerization domain, such as a CH2-CH3 domain, or an optional polypeptide (WO 2005/070966, WO 2006/107786 A WO 2006/107617 A, WO 2007/046893). PCT Publication Nos. WO 2013/174873, WO 2011/133886, and WO 2010/136172 disclose trispecific antibodies in which the CL and CH1 domains are transformed by their respective natural positions, and the VL and VH domains have been diversified ( WO 2008/027236, WO 2010/108127) to allow them to bind more than one antigen. PCT Publication Nos. WO 2013/163427 and WO 2013/119903 disclose modifying a CH2 domain to comprise a fusion protein adduct comprising a binding domain. PCT Publication Nos. WO 2010/028797, WO 2010028796, and WO 2010/028795 disclose recombinant antibodies whose Fc region has been replaced with additional VL and VH domains to form a trivalent binding molecule. PCT Publication Nos. WO 2003/025018 and WO2003012069 disclose recombinant diabody, the single strand of which comprises a scFv domain. PCT Publication No. WO 2013/006544 discloses multivalent Fab molecules which are synthesized as single polypeptide chains and then subjected to proteolysis to produce heterodimerized structures. PCT Publication No. WO 2014/022540, WO 2013/003652, WO 2012/162583, WO 2012/156430, WO 2011/086091, WO 2008/024188, WO 2007/024715, WO 2007/075270, WO 1998/002463, WO 1992 /022583 and WO 1991/003493 disclose the addition of additional binding domains or functional groups to antibodies or antibody portions (eg, addition of a diabody to the light chain of an antibody or addition of additional VL and VH domains to the light chain and heavy of the antibody) Chains or addition of heterologous fusion proteins or linking multiple Fab domains to each other).

本領域已經另外注意到產生在能夠結合兩個或多個不同表位種類(即，除了雙價或多價之外顯示雙特異性或多特異性)方面不同於這樣的天然抗體的雙抗體的能力(見，例如，Holliger等(1993) “’Diabodies’: Small Bivalent And Bispecific Antibody Fragments, ” Proc. Natl. Acad. Sci. (U.S.A.) 90:6444-6448、US 2004/0058400 (Hollinger等)；US 2004/0220388/WO 02/02781 (Mertens等)；Alt等(1999) FEBS Lett. 454(1-2):90-94；Lu, D.等(2005) “A Fully Human Recombinant IgG-Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity ,” J. Biol. Chem. 280(20):19665-19672；WO 02/02781 (Mertens等)；Olafsen, T.等(2004) “Covalent Disulfide-Linked Anti-CEA Diabody Allows Site-Specific Conjugation And Radiolabeling For Tumor Targeting Applications ,” Protein Eng. Des. Sel. 17(1):21-27；Wu, A.等(2001) “Multimerization Of A Chimeric Anti-CD20 Single Chain Fv-Fv Fusion Protein Is Mediated Through Variable Domain Exchange ,” Protein Engineering 14(2):1025-1033；Asano等(2004) “A Diabody For Cancer Immunotherapy And Its Functional Enhancement By Fusion Of Human Fc Domain ,” Abstract 3P-683, J. Biochem. 76(8):992；Takemura, S.等(2000) “Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System, ” Protein Eng. 13(8):583-588；Baeuerle, P.A.等(2009) “Bispecific T-Cell Engaging Antibodies For Cancer Therapy ,” Cancer Res. 69(12):4941-4944)。It has been additionally noted in the art to generate diabodies that differ from such natural antibodies in that they are capable of binding two or more different epitope species (ie, exhibiting bispecificity or multispecificity in addition to bivalent or multivalent). Capabilities (see, for example, Holliger et al. (1993) "'Diabodies': Small Bivalent And Bispecific Antibody Fragments, " Proc. Natl. Acad. Sci. (USA) 90:6444-6448, US 2004/0058400 (Hollinger et al); US 2004/0220388/WO 02/02781 (Mertens et al); Alt et al (1999) FEBS Lett. 454(1-2): 90-94; Lu, D. et al. (2005) “ A Fully Human Recombinant IgG-Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity ," J. Biol. Chem. 280(20): 19665-19672; WO 02/02781 (Mertens et al); Olafsen, T. (2004) “ Covalent Disulfide-Linked Anti-CEA Diabody Allows Site-Specific Conjugation And Radiolabeling For Tumor Targeting Applications ,” Protein Eng. Des. Sel. 17(1): 21-27; Wu, A. et al. (2001) “ Multimerization Of A Chimeric Anti-CD20 Single Chain Fv-Fv Fusion Protein Is Mediated Through Variable Domain Exchange ,” Protein Engineering 14(2): 1025-1033; Asano et al. (2004) “ A Diabody For Cancer Immunotherapy And Its Functional Enhancement By Fusion Of Human Fc Domain ,” Abstract 3P -683, J. Biochem. 76(8): 992; Takemura, S. et al. (2000) “ Configuration Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System, ” Protein Eng. 13(8): 583-588 Baeuerle, PA et al. (2009) " Bispecific T-Cell Engaging Antibodies For Cancer Therapy ," Cancer Res. 69(12): 4941-4944).

雙抗體的設計是基於被稱為單鏈可變結構域片段(scFv)的抗體衍生物。這樣的分子通過利用短連接肽連接輕鏈和/或重鏈可變結構域而製備。B Bird等(1988) (“Single-Chain Antigen-Binding Proteins ,” Science 242:423-426)描述了連接肽的例子，其在一個可變區的羧基末端和另一可變區的氨基末端之間橋接約3.5 nm。已經設計和使用了其他序列的連接體(Bird等(1988) “Single-Chain Antigen-Binding Proteins ,” Science 242:423-426)。連接體進而可被修飾用於另外的功能，比如藥物的附著或附著至固體載體。可重組或合成產生單鏈變體。為了合成產生scFv，可使用自動合成儀。為了重組體產生scFv，可將包含編碼scFv的多核苷酸的合適質粒引入適當的宿主細胞中，所述宿主細胞可以是真核細胞，比如酵母細胞、植物細胞、昆蟲細胞或哺乳動物細胞，或原核細胞，比如大腸埃希氏菌。可通過常規操作比如多核苷酸的連接製備編碼感興趣的scFv的多核苷酸。可使用本領域已知的標準蛋白質純化技術分離所得scFv。The design of the diabody is based on an antibody derivative called a single chain variable domain fragment (scFv). Such molecules are prepared by ligating light chain and/or heavy chain variable domains with short linker peptides. B Bird et al. (1988) (" Single-Chain Antigen-Binding Proteins ," Science 242:423-426) describes examples of linker peptides that are at the carboxy terminus of one variable region and the amino terminus of another variable region. The bridge is approximately 3.5 nm. Other sequences of adaptors have been designed and used (Bird et al. (1988) " Single-Chain Antigen-Binding Proteins ," Science 242: 423-426). The linker can in turn be modified for additional functions, such as attachment or attachment of a drug to a solid support. Recombinant or synthetic can produce single-chain variants. For the synthesis to produce scFv, an automated synthesizer can be used. For recombinant production of scFv, a suitable plasmid comprising a polynucleotide encoding an scFv can be introduced into a suitable host cell, such as a yeast cell, a plant cell, an insect cell or a mammalian cell, or Prokaryotic cells, such as Escherichia coli. Polynucleotides encoding the scFv of interest can be prepared by conventional manipulations such as ligation of polynucleotides. The resulting scFv can be isolated using standard protein purification techniques known in the art.

雙特異性結合分子(例如，非單特異性雙抗體)的供應提供相比抗體的明顯優勢，包括但不限於足以共連接和/或共定位表達不同表位的不同細胞的“反式(trans)”結合能力和/或足以共連接和/或共定位由相同細胞表達的不同分子的“順式(cis)”結合能力。因此，雙特異性結合分子(例如，非單特異性雙抗體)具有廣泛的應用，包括治療和免疫診斷。在各種應用中，雙特異性在設計和工程化雙抗體方面允許大的靈活性，提供對多聚抗原的增強的親合力、不同抗原的交聯和對特定細胞類型的導向靶向，這取決於兩個靶抗原的存在。由於其增加的價、低離解速率和從迴圈中快速清除(對於小尺寸的雙抗體，為~50 kDa或以下)，本領域中已知的雙抗體分子在腫瘤成像領域還顯示特別的用途(Fitzgerald等(1997)“Improved Tumor Targeting By Disulphide Stabilized Diabodies Expressed In Pichia pastoris, ” Protein Eng. 10: 1221-1225)。The supply of bispecific binding molecules (eg, non-monospecific diabodies) provides significant advantages over antibodies, including but not limited to "trans (trans) sufficient to co-ligate and/or co-localize different cells expressing different epitopes "binding ability and/or "cis" binding ability sufficient to co-ligate and/or co-localize different molecules expressed by the same cell. Thus, bispecific binding molecules (eg, non-monospecific diabodies) have a wide range of applications, including therapeutic and immunodiagnostic. In various applications, bispecificity allows for great flexibility in designing and engineering diabody, providing enhanced affinity for multimeric antigens, cross-linking of different antigens, and targeted targeting of specific cell types, depending on The presence of two target antigens. Due to its increased valency, low dissociation rate, and rapid clearance from the loop (~50 kDa or less for small size diabody), diabody molecules known in the art also show particular use in the field of tumor imaging. (Fitzgerald et al. (1997) "Improved Tumor Targeting By Disulphide Stabilized Diabodies Expressed In Pichia pastoris," Protein Eng. 10: 1221-1225).

產生雙特異性雙抗體的能力引起它們(以“反式”) 將兩個細胞共連接在一起的用途，例如，通過共連接在不同的細胞表面上存在的受體(例如，將細胞毒性T-細胞與腫瘤細胞交聯) (Staerz等(1985)“Hybrid Antibodies Can Target Sites For Attack By T Cells,” Nature 314:628-631；和Holliger等(1996)“Specific Killing Of LymphomaCells By Cytotoxic T-Cells Mediated By A Bispecific Diabody,” Protein Eng. 9:299-305；Marvin等(2005) “Recombinant Approaches To IgG-Like Bispecific Antibodies ,” Acta Pharmacol. Sin. 26:649-658)。可選地(或另外)，雙特異性(或三特異性或多特異性)雙抗體可用於(以“順式”)共連接存在於相同細胞表面上的分子，比如受體等。共連接不同的細胞和/或受體可用於調節效應子功能和/或免疫細胞信號傳導。包括表位-結合位點的多特異性分子(例如雙特異性雙抗體)可被引導至任何在T淋巴細胞、自然殺傷(NK)細胞、抗原呈遞細胞或其他單核細胞上表達的免疫細胞的表面決定簇，諸如CD2、CD3、CD8、CD16、T細胞受體(TCR)、NKG2D等。尤其地，針對存在於免疫效應細胞上的細胞表面受體的表位-結合位點可用於產生能夠介導重定向細胞殺傷的多特異性結合分子。The ability to generate bispecific diabodies causes their use ("trans") to co-ligate two cells together, for example, by co-ligating receptors present on different cell surfaces (eg, cytotoxic T) - cell cross-linking with tumor cells) (Staerz et al. (1985) "Hybrid Antibodies Can Target Sites For Attack By T Cells," Nature 314: 628-631; and Holliger et al. (1996) "Specific Killing Of Lymphoma Cells By Cytotoxic T- Cells Mediated By A Bispecific Diabody," Protein Eng. 9: 299-305; Marvin et al. (2005) " Recombinant Approaches To IgG-Like Bispecific Antibodies ," Acta Pharmacol. Sin. 26: 649-658). Alternatively (or in addition), bispecific (or trispecific or multispecific) diabodyes can be used (by "cis") to co-ligate molecules present on the same cell surface, such as receptors and the like. Co-ligation of different cells and/or receptors can be used to modulate effector function and/or immune cell signaling. Multispecific molecules including epitope-binding sites (eg, bispecific diabodies) can be directed to any immune cell expressed on T lymphocytes, natural killer (NK) cells, antigen presenting cells, or other monocytes. Surface determinants such as CD2, CD3, CD8, CD16, T cell receptor (TCR), NKG2D, and the like. In particular, epitope-binding sites directed against cell surface receptors present on immune effector cells can be used to generate multispecific binding molecules capable of mediating redirected cell killing.

然而，上述優勢需要突出的成本。這類非單特異性雙抗體的形成需要成功組裝兩個或更多個獨特和不同的多肽(即，這樣的形成需要通過不同多肽鏈種類的異源二聚化而形成雙抗體)。該事實與單特異性雙抗體不同，其通過一致的多肽鏈的同源二聚化而形成。由於至少兩個不同的多肽(即，兩個多肽種類)必須被提供以形成非單特異性雙抗體並且由於這樣的多肽的同源二聚化導致失活分子(Takemura, S.等(2000) “Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System, ” Protein Eng. 13(8):583-588)，這樣的多肽的產生必須以防止相同種類的多肽之間的共價結合的方式完成(即，以便防止同源二聚化) (Takemura, S.等(2000) “Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System, ” Protein Eng. 13(8):583-588)。所以，現有技術教導了這類多肽的非共價締合(見，例如，Olafsen等(2004)“Covalent Disulfide-Linked Anti-CEA Diabody Allows Site-Specific Conjugation And Radiolabeling For Tumor Targeting Applications,” Prot. Engr. Des. Sel. 17:21-27；Asano等(2004) “A Diabody For Cancer Immunotherapy And Its Functional Enhancement By Fusion Of Human Fc Domain ,” Abstract 3P-683, J. Biochem. 76(8):992；Takemura, S.等(2000) “Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System, ” Protein Eng. 13(8):583-588；Lu, D.等( 2005) “A Fully Human Recombinant IgG-Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity ,” J. Biol. Chem. 280(20):19665-19672)。However, the above advantages require outstanding costs. The formation of such non-monospecific diabody requires successful assembly of two or more unique and distinct polypeptides (ie, such formation requires heterodimerization of different polypeptide chain species to form a diabody). This fact is different from monospecific diabody, which is formed by homodimerization of a consistent polypeptide chain. Since at least two different polypeptides (ie, two polypeptide species) must be provided to form a non-monospecific diabodies and inactivated molecules due to homodimerization of such polypeptides (Takemura, S. et al. (2000) " Configuration Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System, " Protein Eng. 13(8): 583-588), the production of such polypeptides must be in a manner that prevents covalent binding between polypeptides of the same species. Completed (ie, to prevent homodimerization) (Takemura, S. et al. (2000) " Configuration Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System, " Protein Eng. 13(8): 583-588) . Therefore, the prior art teaches non-covalent association of such polypeptides (see, for example, Olafsen et al. (2004) "Covalent Disulfide-Linked Anti-CEA Diabody Allows Site-Specific Conjugation And Radiolabeling For Tumor Targeting Applications," Prot. Engr Des. Sel. 17:21-27; Asano et al. (2004) " A Diabody For Cancer Immunotherapy And Its Functional Enhancement By Fusion Of Human Fc Domain ," Abstract 3P-683, J. Biochem. 76(8):992; Takemura, S. et al. (2000) “ Configuration Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System, ” Protein Eng. 13(8): 583-588; Lu, D. et al. ( 2005) “ A Fully Human Recombinant IgG-Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity , "J. Biol. Chem. 280(20): 19665-19672).

然而，本領域已經認識到由非共價締合的多肽組成的雙特異性雙抗體不穩定並且容易解離成非功能單體(見，例如，Lu, D.等(2005) “A Fully Human RecombinantIgG-Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity ,” J. Biol. Chem. 280(20):19665-19672)。However, it has been recognized in the art that bispecific diabodies consisting of non-covalently associated polypeptides are unstable and readily dissociable into non-functional monomers (see, eg, Lu, D. et al. (2005) " A Fully Human Recombinant IgG-Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity , "J. Biol. Chem. 280(20): 19665-19672).

面對這樣的挑戰，本領域已經成功地開發了穩定的、共價結合的異源二聚非單特異性雙抗體，稱為DART® ( 雙親和力重靶向試劑 )雙抗體；見，例如，美國專利公開號2013-0295121、2010-0174053和2009-0060910；歐洲專利公開號EP 2714079、EP 2601216、EP 2376109、EP 2158221；和PCT公開號WO 2012/162068、WO 2012/018687、WO 2010/080538；和Sloan, D.D.等(2015) “Targeting HIV Reservoir in Infected CD4 T Cells by Dual-Affinity Re-targeting Molecules (DARTs) that Bind HIV Envelope and Recruit Cytotoxic T Cells ,” PLoS Pathog. 11(11):e1005233. doi: 10.1371/journal.ppat.1005233；Al Hussaini, M.等(2015) “Targeting CD123 In AML Using A T-Cell Directed Dual-Affinity Re-Targeting (DART®) Platform ,” Blood pii: blood-2014-05-575704；Chichili, G.R.等(2015) “A CD3xCD123 Bispecific DART For Redirecting Host T Cells To Myelogenous Leukemia: Preclinical Activity And Safety In Nonhuman Primates ,” Sci. Transl. Med. 7(289):289ra82；Moore, P.A.等(2011) “Application Of Dual Affinity Retargeting Molecules To Achieve Optimal Redirected T-Cell Killing Of B-Cell Lymphoma ,” Blood 117(17):4542-4551；Veri, M.C.等(2010) “Therapeutic Control Of B Cell Activation Via Recruitment Of Fcgamma Receptor IIb (CD32B) Inhibitory Function With A Novel Bispecific Antibody Scaffold ,” Arthritis Rheum. 62(7):1933-1943；Johnson, S.等(2010) “Effector Cell Recruitment With Novel Fv-Based Dual-Affinity Re-Targeting Protein Leads To Potent Tumor Cytolysis And in vivo B-Cell Depletion ,” J. Mol. Biol. 399(3):436-449)。這樣的雙抗體包括兩條或多條共價複合的多肽並涉及將一個或多個半胱氨酸殘基工程化到每個應用的多肽種類中，其允許形成二硫鍵，從而將一對或多對這類多肽鏈彼此共價結合。例如，將半胱氨酸殘基添加至這樣的構建體的C-末端已經被證明允許涉及的多肽鏈之間的二硫結合，穩定了產生的雙抗體，而不干擾雙抗體的結合特性。Faced with such challenges, stable, covalently bound heterodimeric non-monospecific diabodies, known as DART® ( bi- parent retargeting agents ) diabody, have been successfully developed in the art; see, for example, US Patent Publication Nos. 2013-0295121, 2010-0174053, and 2009-0060910; European Patent Publication No. EP 2714079, EP 2601216, EP 2376109, EP 2158221; and PCT Publication No. WO 2012/162068, WO 2012/018687, WO 2010/080538 And Sloan, DD et al. (2015) “ Targeting HIV Reservoir in Infected CD4 T Cells by Dual-Affinity Re-targeting Molecules (DARTs) that Bind HIV Envelope and Recruit Cytotoxic T Cells ,” PLoS Pathog. 11(11):e1005233. Doi: 10.1371/journal.ppat.1005233; Al Hussaini, M. et al. (2015) “ Targeting CD123 In AML Using A T-Cell Directed Dual-Affinity Re-Targeting (DART®) Platform ,” Blood pii: blood-2014- 05-575704; Chichili, GR et al. (2015) “ A CD3xCD123 Bispecific DART For Redirecting Host T Cells To Myelogenous Leukemia: Preclinical Activity And Safety In Nonhuman Primates ,” Sci. Transl. Med. 7(289): 289ra8 2; Moore, PA, et al. (2011) “ Application Of Dual Affinity Retargeting Molecules To Achieve Optimal Redirected T-Cell Killing Of B-Cell Lymphoma ,” Blood 117(17): 4542-4551; Veri, MC et al. (2010) “ Therapeutic Control Of B Cell Activation Via Recruitment Of Fcgamma Receptor IIb (CD32B) Inhibitory Function With A Novel Bispecific Antibody Scaffold ," Arthritis Rheum. 62(7): 1933-1943; Johnson, S. et al. (2010) " Effector Cell Recruitment With Novel Fv-Based Dual-Affinity Re-Targeting Protein Leads To Potent Tumor Cytolysis And in vivo B-Cell Depletion ," J. Mol. Biol. 399(3): 436-449). Such diabody includes two or more covalently complexed polypeptides and is involved in engineering one or more cysteine residues into the polypeptide species of each application, which allows the formation of disulfide bonds, thereby Or a plurality of such polypeptide chains are covalently bound to each other. For example, the addition of a cysteine residue to the C-terminus of such a construct has been shown to allow for disulfide binding between the polypeptide chains involved, stabilizing the diabody produced without interfering with the binding properties of the diabody.

已經描述了這類分子的許多變型(見，例如，美國專利公開號2015/0175697、2014/0255407、2014/0099318、2013/0295121、2010/0174053；2009/0060910；2007-0004909；歐洲專利公開號EP 2714079、EP 2601216、EP 2376109、EP 2158221、EP 1868650；和PCT公開號WO 2012/162068、WO 2012/018687、WO 2010/080538、WO 2006/113665)，並且提供在本文中。A number of variations of such molecules have been described (see, for example, U.S. Patent Publication Nos. 2015/0175697, 2014/0255407, 2014/0099318, 2013/0295121, 2010/0174053; 2009/0060910; 2007-0004909; European Patent Publication No. EP 2 714 079, EP 2 601 216, EP 2 376 109, EP 2 158 221, EP 1 868 650; and PCT Publication Nos. WO 2012/162068, WO 2012/018687, WO 2010/080538, WO 2006/113665), and hereby incorporated herein.

用於期望四價分子而不需要Fc的應用的可選的構建體在本領域中是已知的，包括但不限於四價串聯抗體，也稱為“TandAbs ”(見，例如美國專利公開號2005-0079170、2007-0031436、2010-0099853、2011-020667 2013-0189263；歐洲專利公開號EP 1078004、EP 2371866、EP 2361936和EP 1293514；PCT公開號WO 1999/057150、WO 2003/025018，和WO 2013/013700)，其通過每個具有VH1、VL2、VH2和VL2結構域的兩條相同多肽鏈的同源二聚化而形成。Alternative constructs for use in applications where a tetravalent molecule is desired without the need for Fc are known in the art, including but not limited to tetravalent tandem antibodies, also referred to as " TandAbs " (see, e.g., U.S. Patent Publication No. 2005-0079170, 2007-0031436, 2010-0099853, 2011-020667 2013-0189263; European Patent Publication No. EP 1078004, EP 2371866, EP 2361936 and EP 1293514; PCT Publication No. WO 1999/057150, WO 2003/025018, and WO 2013/013700), which is formed by homodimerization of two identical polypeptide chains each having the VH1, VL2, VH2 and VL2 domains.

最近，已經描述了併入兩個雙抗體型結合結構域和一個非雙抗體型結構域和Fc區域的三價結構(見例如PCT公開號WO 2015/184207和WO 2015/184203)。這類三價結合分子可用於生成單特異性、雙特異性或三特異性分子。結合三種不同表位的能力提供增強的性能。圖 6A-6F 提供了包括3或4條多肽鏈的這類三價結合分子的示意圖。Recently, trivalent structures incorporating two diabody-type binding domains and one non-diabody-type domain and Fc region have been described (see, for example, PCT Publication Nos. WO 2015/184207 and WO 2015/184203). Such trivalent binding molecules can be used to generate monospecific, bispecific or trispecific molecules. The ability to combine three different epitopes provides enhanced performance. Figures 6A-6F provide schematic representations of such trivalent binding molecules comprising 3 or 4 polypeptide chains.

IVIV .. 優化的抗Optimized resistance -ROR1-ROR1 可變結構域Variable domain

本發明的優選的優化的ROR1-結合分子包括能夠結合人ROR1的連續或不連續(例如，構象的)表位的抗體、雙抗體、BiTEs、三價結合分子等。本發明的優化的ROR1-結合分子還優選地顯示結合一種或多種非人物種，尤其是非人靈長類物種(例如食蟹猴、黑猩猩、獼猴(macaque)等)的ROR1分子的能力。人ROR1多肽的代表性長等同型(NCBI序列NP_005003.2，包括29個氨基酸殘基信號序列，以底線表示)(SEQ ID NO:5 )是： MHRPRRRGTR PPLLALLAAL LLAARGAAA Q ETELSVSAEL VPTSSWNISS ELNKDSYLTL DEPMNNITTS LGQTAELHCK VSGNPPPTIR WFKNDAPVVQ EPRRLSFRST IYGSRLRIRN LDTTDTGYFQ CVATNGKEVV SSTGVLFVKF GPPPTASPGY SDEYEEDGFC QPYRGIACAR FIGNRTVYME SLHMQGEIEN QITAAFTMIG TSSHLSDKCS QFAIPSLCHY AFPYCDETSS VPKPRDLCRD ECEILENVLC QTEYIFARSN PMILMRLKLP NCEDLPQPES PEAANCIRIG IPMADPINKN HKCYNSTGVD YRGTVSVTKS GRQCQPWNSQ YPHTHTFTAL RFPELNGGHS YCRNPGNQKE APWCFTLDEN FKSDLCDIPA CDSKDSKEKN KMEILYILVP SVAIPLAIAL LFFFICVCRN NQKSSSAPVQ RQPKHVRGQN VEMSMLNAYK PKSKAKELPL SAVRFMEELG ECAFGKIYKG HLYLPGMDHA QLVAIKTLKD YNNPQQWTEF QQEASLMAEL HHPNIVCLLG AVTQEQPVCM LFEYINQGDL HEFLIMRSPH SDVGCSSDED GTVKSSLDHG DFLHIAIQIA AGMEYLSSHF FVHKDLAARN ILIGEQLHVK ISDLGLSREI YSADYYRVQS KSLLPIRWMP PEAIMYGKFS SDSDIWSFGV VLWEIFSFGL QPYYGFSNQE VIEMVRKRQL LPCSEDCPPR MYSLMTECWN EIPSRRPRFK DIHVRLRSWE GLSSHTSSTT PSGGNATTQT TSLSASPVSN LSNPRYPNYM FPSQGITPQG QIAGFIGPPI PQNQRFIPIN GYPIPPGYAA FPAAHYQPTG PPRVIQHCPP PKSRSPSSAS GSTSTGHVTS LPSSGSNQEA NIPLLPHMSI PNHPGGMGIT VFGNKSQKPY KIDSKQASLL GDANIHGHTE SMISAELPreferred optimized ROR1-binding molecules of the invention include antibodies, diabodies, BiTEs, trivalent binding molecules, and the like that are capable of binding to a continuous or discontinuous (e.g., conformational) epitope of human ROR1. The optimized ROR1-binding molecules of the invention also preferably exhibit the ability to bind to one or more non-human species, particularly ROR1 molecules of non-human primate species (eg, cynomolgus, chimpanzee, macaque, etc.). A representative long isoform of the human ROR1 polypeptide (NCBI sequence NP_005003.2, including a 29 amino acid residue signal sequence, indicated by the bottom line) ( SEQ ID NO: 5 ) is: MHRPRRRGTR PPLLALLAAL LLAARGAAA Q ETELSVSAEL VPTSSWNISS ELNKDSYLTL DEPMNNITTS LGQTAELHCK VSGNPPPTIR WFKNDAPVVQ EPRRLSFRST IYGSRLRIRN LDTTDTGYFQ CVATNGKEVV SSTGVLFVKF GPPPTASPGY SDEYEEDGFC QPYRGIACAR FIGNRTVYME SLHMQGEIEN QITAAFTMIG TSSHLSDKCS QFAIPSLCHY AFPYCDETSS VPKPRDLCRD ECEILENVLC QTEYIFARSN PMILMRLKLP NCEDLPQPES PEAANCIRIG IPMADPINKN HKCYNSTGVD YRGTVSVTKS GRQCQPWNSQ YPHTHTFTAL RFPELNGGHS YCRNPGNQKE APWCFTLDEN FKSDLCDIPA CDSKDSKEKN KMEILYILVP SVAIPLAIAL LFFFICVCRN NQKSSSAPVQ RQPKHVRGQN VEMSMLNAYK PKSKAKELPL SAVRFMEELG ECAFGKIYKG HLYLPGMDHA QLVAIKTLKD YNNPQQWTEF QQEASLMAEL HHPNIVCLLG AVTQEQPVCM LFEYINQGDL HEFLIMRSPH SDVGCSSDED GTVKSSLDHG DFLHIAIQIA AGMEYLSSHF FVHKDLAARN ILIGEQLHVK ISDLGLSREI YSADYYRVQS KSLLPIRWMP PEAIMYGKFS SDSDIWSFGV VLWEIFSFGL QPYYGFSNQE VIEMVRKRQL LPCSEDCPPR MYSLMTECWN EIPSRRPRFK DIHVRLRSWE GLSSHTSSTT PSGGNATTQT TSLSASPVSN LSNPRYPNYM FPSQGITPQG QIAGFIGPPI PQNQRFIPIN GYPIPPGYAA FPAAHYQPTG PPRVIQHCPP PKSRSPSSAS GSTSTGHVTS LPSSGSNQEA NIPLLPHMSI PNHPGGMGIT VFGNKSQKPY KIDSKQASLL GDANIHGHTE SMISAEL

在ROR1(SEQ ID NO:5 )的937個氨基酸殘基中，殘基1-29是信號序列，殘基30-406是細胞外結構域，殘基407-427是跨膜結構域，殘基428- 937是細胞質結構域。已知幾種等同型和天然變體。Among the 937 amino acid residues of ROR1 ( SEQ ID NO: 5 ), residues 1-29 are signal sequences, residues 30-406 are extracellular domains, and residues 407-427 are transmembrane domains, residues 428-937 is a cytoplasmic domain. Several equivalent and natural variants are known.

本發明尤其包括包含免疫特異性結合人ROR1多肽的表位的優化的抗-ROR1可變結構域(即VL和/或VH結構域)的ROR1結合分子(例如抗體、雙抗體、三價結合分子等)。如本文所使用的，這樣的ROR1可變結構域分別被稱為“抗-ROR1-VL”和“抗-ROR1-VH”。The invention particularly includes ROR1 binding molecules (eg, antibodies, diabody, trivalent binding molecules) comprising an optimized anti-ROR1 variable domain (ie, a VL and/or VH domain) that immunospecifically binds to an epitope of a human ROR1 polypeptide. Wait). As used herein, such ROR1 variable domains are referred to as "anti-ROR1-VL" and "anti-ROR1-VH," respectively.

本發明的ROR1-結合分子尤其包括具有優化的抗-ROR1-VL結構域和/或抗-ROR1-VH結構域的分子，其免疫特異性結合人ROR1多肽，尤其是包括SEQ ID NO:5 的殘基30-406的人ROR1多肽，的表位。優選地，與包括非優化的親本抗-ROR1-VL結構域和抗-ROR1-VH結構域的ROR1-結合分子相比，這類優化的ROR1-結合分子表現出對於人ROR1增強的結合親和力，和/或是去免疫化的，以降低這類分子的免疫原性。更優選地，本發明涉及優化的ROR1-結合分子，其顯示對ROR1增強的結合親和力和降低的免疫原性。The ROR1-binding molecules of the invention include, inter alia, molecules having an optimized anti-ROR1-VL domain and/or an anti-ROR1-VH domain that immunospecifically bind to a human ROR1 polypeptide, particularly comprising SEQ ID NO: The epitope of the human ROR1 polypeptide of residues 30-406. Preferably, such optimized ROR1-binding molecules exhibit enhanced binding affinity for human ROR1 compared to ROR1-binding molecules including a non-optimized parental anti-ROR1-VL domain and an anti-ROR1-VH domain. , and / or deimmunized to reduce the immunogenicity of such molecules. More preferably, the invention relates to an optimized ROR1-binding molecule which exhibits enhanced binding affinity to ROR1 and reduced immunogenicity.

下麵和圖 7A 提供了親本抗-ROR1-VL結構域(SEQ ID NO:6 )的氨基酸序列，CDR_L 殘基以底線表示。 QLVLTQSPSA SASLGSSVKL TC TLSSGHKT DTID WYQQQP GKAPRYLMK L EGSGSYNKGS GVPDRFGSGS SSGADRYLTI SSLQSEDEAD YYC GTDYPGN YL FGGGTQLT VLGThe amino acid sequence of the parent anti-ROR1-VL domain ( SEQ ID NO: 6 ) is provided below and in Figure 7A , the CDR _L residues are indicated by the bottom line. QLVLTQSPSA SASLGSSVKL TC TLSSGHKT DTID WYQQQP GKAPRYLMK L EGSGSYNKGS GVPDRFGSGS SSGADRYLTI SSLQSEDEAD YYC GTDYPGN YL FGGGTQLT VLG

下麵和圖 7B 提供了親本抗-ROR1-VH結構域(SEQ ID NO:7 )的氨基酸序列，CDR_H 殘基以底線表示。 QEQLVESGGG LVQPGGSLRL SCAASGFTFS DYYMS WVRQA PGKGLEWVA T IYPSSGKTYY ADSVKG RFTI SSDNAKNSLY LQMNSLRAED TAVYYCAR DS YADDAALFDI WGQGTTVTVS SThe amino acid sequence of the parent anti-ROR1-VH domain ( SEQ ID NO: 7 ) is provided below and in Figure 7B , the CDR _H residues are indicated by the bottom line. QEQLVESGGG LVQPGGSLRL SCAASGFTFS DYYMS WVRQA PGKGLEWVA T IYPSSGKTYY ADSVKG RFTI SSDNAKNSLY LQMNSLRAED TAVYYCAR DS YADDAALFDI WGQGTTVTVS S

在某些實施方案中，包括本發明的優化的抗-ROR1-VL和/或VH結構域的ROR1-結合分子(例如scFv、抗體、雙特異性雙抗體等)的特徵在於以下標準中的任何一個、兩個、三個、四個、五個、六個、七個、八個或九個： (1) 免疫特異性地結合在癌細胞表面上內源性表達的人ROR1的能力； (2) 相對於包括親本抗-ROR1-VL和抗-ROR1-VH結構域的ROR1-結合分子，以增強的結合親和力免疫特異性地結合人ROR1的能力； (3) 相比包括親本抗-ROR1-VL和抗-ROR1-VH結構域的ROR1-結合分子的單價平衡結合常數(K_D )，以較低的單價平衡結合常數(K_D )免疫特異性地結合人ROR1的能力； (4) 相比包括親本抗-ROR1-VL和抗-ROR1-VH結構域的ROR1-結合分子的單價平衡結合常數(K_D )，以其二分之一的單價平衡結合常數(K_D )免疫特異性地結合人ROR1的能力； (5) 相比包括親本抗-ROR1-VL和抗-ROR1-VH結構域的ROR1-結合分子的單價締合速率(K_a )，以較高的單價締合速率(K_a )免疫特異性地結合人ROR1的能力； (6) 相比包括親本抗-ROR1-VL和抗-ROR1-VH結構域的ROR1-結合分子的單價解離速率(K_d )，以較低的單價解離速率(K_d )免疫特異性地結合人ROR1的能力； (7) 免疫特異性地結合非人靈長類動物ROR1(例如食蟹猴的ROR1)的能力； (8) 相對於包括親本抗-ROR1-VL和抗-ROR1-VH結構域的ROR1-結合分子的免疫原性，降低的免疫原性；和/或 (9) 相對於包括親本抗-ROR1-VL和抗-ROR1-VH結構域的ROR1-結合分子的介導重定向細胞殺傷的能力(如果有的話)，增強的介導重定向細胞殺傷的能力。In certain embodiments, ROR1-binding molecules (eg, scFv, antibodies, bispecific diabodies, etc.) comprising an optimized anti-ROR1-VL and/or VH domain of the invention are characterized by any of the following criteria One, two, three, four, five, six, seven, eight or nine: (1) the ability to immunospecifically bind to human ROR1 that is endogenously expressed on the surface of cancer cells; 2) the ability to immunospecifically bind to human ROR1 with enhanced binding affinity relative to ROR1-binding molecules comprising the parental anti-ROR1-VL and anti-ROR1-VH domains; (3) compared to including parental antibodies Unit -ROR1-VL, and anti-binding molecule ROR1- -ROR1-VH domains of the equilibrium binding constant (K _D), a monovalent lower equilibrium binding constant (K _D) the ability to immunospecifically bind to a human ROR1; ( 4) compared to the parental anti comprising -ROR1-VL ROR1- binding molecule and an anti--ROR1-VH domains monovalent equilibrium binding constant (K _D), one half of its monovalent equilibrium binding constant (K _D) The ability to immunospecifically bind to human ROR1; (5) ROR1-binding molecules compared to the parental anti-ROR1-VL and anti-ROR1-VH domains Monovalent association rate (K _a), the monovalent higher association rate (K _a) the ability to immunospecifically bind to a human ROR1; (6) as compared to the parental anti comprising -ROR1-VL -ROR1-VH and anti- The monovalent dissociation rate (K _d ) of the ROR1-binding molecule of the domain, the ability to immunospecifically bind to human ROR1 at a lower rate of monovalent dissociation (K _d ); (7) immunospecific binding of non-human primates The ability of animal ROR1 (eg, ROR1 of cynomolgus monkeys); (8) reduced immunogenicity relative to the immunogenicity of ROR1-binding molecules including the parental anti-ROR1-VL and anti-ROR1-VH domains; And/or (9) an enhanced mediated weight that mediates the ability to redirect cell killing, if any, relative to a ROR1-binding molecule comprising the parental anti-ROR1-VL and anti-ROR1-VH domains The ability to direct cell killing.

如本文別處所述，可以使用表面等離子體共振，例如通過BIACORE®分析來測定ROR1-結合分子的結合常數。表面等離子體共振資料可以擬合到1:1朗繆爾結合模型(Langmuir binding model)(同步的ka kd)和從速率常數kd/ka的比率計算的平衡結合常數K_D 。這類結合常數可以針對單價ROR1-結合分子(即，包括單個ROR1表位-結合位點的分子)、二價ROR1-結合分子(即，包括兩個ROR1表位-結合位點的分子)或具有更高價的ROR1-結合分子(例如，包括三個、四個或更多個ROR1表位-結合位點的分子)被測定。As described elsewhere herein, surface plasmon resonance can be used, for example, by BIACORE® analysis to determine the binding constant of a ROR1-binding molecule. The surface plasmon resonance data can be fitted to the 1:1 Langmuir binding model (synchronized ka kd) and the equilibrium binding constant K _D calculated from the ratio of the rate constant kd/ka. Such binding constants can be directed to a monovalent ROR1-binding molecule (ie, a molecule comprising a single ROR1 epitope-binding site), a bivalent ROR1-binding molecule (ie, a molecule comprising two ROR1 epitope-binding sites) or A ROR1-binding molecule having a higher valency (for example, a molecule including three, four or more ROR1 epitope-binding sites) is determined.

如本文所使用的，術語“重定向細胞殺傷 (redirected cell killing )”是指分子通過結合存在於效應細胞和靶細胞表面上的表位將這類免疫效應細胞(例如，T細胞、NK細胞)定位在這類靶細胞的位置，來介導對靶細胞(例如癌細胞)的殺傷的能力，結果導致對靶細胞的殺傷。可以使用細胞毒性T淋巴細胞(CTL)試驗來測定ROR1-結合分子(例如雙特異性ROR1 x CD3-結合分子)介導重定向細胞殺傷活性的能力。這樣的試驗是本領域熟知的，並且優選的試驗在下面描述。As used herein, the term "cell killing redirected (redirected cell killing)" refers to a molecule by binding to an epitope present on the surface of target cells and effector cells this type of immune effector cells (e.g., T cells, NK cells) Positioning at the location of such target cells to mediate the ability to kill target cells, such as cancer cells, results in killing of target cells. A cytotoxic T lymphocyte (CTL) assay can be used to determine the ability of a ROR1-binding molecule (eg, a bispecific ROR1 x CD3-binding molecule) to mediate redirecting cell killing activity. Such tests are well known in the art and preferred assays are described below.

本發明的ROR1-結合分子包括優化的抗-ROR1-VL和/或抗-ROR1-VH結構域。在優選的實施方案中，ROR1-結合分子包括優化的抗-ROR1-VL結構域或優化的抗-ROR1-VH結構域。在更優選的實施方案中，本發明的ROR1-結合分子包括優化的抗-ROR1-VL結構域和優化的抗-ROR1-VH結構域。The ROR1-binding molecules of the invention include optimized anti-ROR1-VL and/or anti-ROR1-VH domains. In a preferred embodiment, the ROR1-binding molecule comprises an optimized anti-ROR1-VL domain or an optimized anti-ROR1-VH domain. In a more preferred embodiment, the ROR1-binding molecules of the invention comprise an optimized anti-ROR1-VL domain and an optimized anti-ROR1-VH domain.

本發明的優選的優化的抗-ROR1-VL結構域的氨基酸序列是SEQ ID NO:6 的變體，並且由SEQ ID NO:8 表示(CDR_L 殘基以底線表示)： QLVLTQSPSA SASLGX₁ SVX₂ L TC TLSSGHKT DTID WYQQQP GKAPRYLMX₃ L EGSGSYNKGS GVPDRFX₄ SGX₅ SSGADX₆ YLTI SSLQSEDEAD YYC GTDX₇ PGN YL FGGGTQLT VLG 其中：X₁ 、X₂ 、X₃ 、X₄ 、X₅ 、X₆ 和X₇ 被獨立地選擇，並且其中：X₁ 為S或G，X₂ 為K、I或N，X₃ 為K或N，X₄ 為G或不存在，X₅ 為S或I，X₆ 為R或W，並且X₇ 為Y或N。The preferred optimized anti-ROR1-VL domain amino acid sequence of the invention is a variant of SEQ ID NO: 6 and is represented by SEQ ID NO: 8 (CDR _L residues are indicated by the bottom line): QLVLTQSPSA SASLG X ₁ SV X ₂ L TC TLSSGHKT DTID WYQQQP GKAPRYLM X ₃ L EGSGSYNKGS GVPDRF X ₄ SG X ₅ SSGAD X ₆ YLTI SSLQSEDEAD YYC GTDX ₇ PGN YL FGGGTQLT VLG Where: X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , X ₆ and X ₇ is independently selected, and wherein: X ₁ is S or G, X ₂ is K, I or N, X ₃ is K or N, X ₄ is G or absent, and X ₅ is S or I, X ₆ Is R or W, and X ₇ is Y or N.

在優選的實施方案中，本發明的ROR1-結合分子包括具有SEQ ID NO:8 的氨基酸序列的優化的抗-ROR1-VL結構域，其中X₆ 為W。In a preferred embodiment, the ROR1-binding molecule of the invention comprises an optimized anti-ROR1-VL domain having the amino acid sequence of SEQ ID NO: 8 , wherein X ₆ is W.

在另一個實施方案中，本發明的優化的ROR1-結合分子包括具有SEQ ID NO:8 的氨基酸序列的優化的抗-ROR1-VL結構域，其中X₆ 為W，並且其中： (a)X₁ 為S或G，X₂ 為K、I或N，X₃ 為K或N，X₄ 為G或不存在，X₅ 為S或I，X₇ 為Y或N； (b)X₁ 為S，X₂ 為K，X₃ 為K，X₄ 為G或不存在，X₅ 為S和X₇ 為N； (c)X₁ 為S，X₂ 為K，X₃ 為K，X₄ 為G或不存在，X₅ 為I和X₇ 為Y； (d)X₁ 為S，X₂ 為K，X₃ 為K，X₄ 為G或不存在，X₅ 為I和X₇ 為N；或 (e)X₁ 為S，X₂ 為K，X₃ 為K，X₄ 為G或不存在，X₅ 為S和X₇ 為Y。In another embodiment, an optimized ROR1-binding molecule of the invention comprises an optimized anti-ROR1-VL domain having the amino acid sequence of SEQ ID NO: 8 , wherein X ₆ is W, and wherein: (a) X ₁ is S or G, X ₂ is K, I or N, X ₃ is K or N, X ₄ is G or absent, X ₅ is S or I, X ₇ is Y or N; (b) X ₁ is S, X ₂ is K, X ₃ is K, X ₄ is G or absent, X ₅ is S and X ₇ is N; (c) X ₁ is S, X ₂ is K, X ₃ is K, X ₄ Is G or absent, X ₅ is I and X ₇ is Y; (d) X ₁ is S, X ₂ is K, X ₃ is K, X ₄ is G or absent, and X ₅ is I and X ₇ is N; or (e) X ₁ is S, X ₂ is K, X ₃ is K, X ₄ is G or absent, X ₅ is S and X ₇ is Y.

本發明的優選的優化的抗-ROR1-VH結構域的氨基酸序列是SEQ ID NO:7 的變體，並且由SEQ ID NO:9 表示(CDR_H 殘基以底線表示)： QEQLVESGGG LVQPGGSLRLSCAASGFTFS DYYMS WX₁ RQA PGKGLEWVAT IYPSSGKTYY ADSX₂ KG RX₃ TI SSDNAKX₄ SLY LQMNSLRAED TAVYYCX₅ R DS YADDAALFDI WGQGTTVTVS S 其中：X₁ 、X₂ 、X₃ 、X₄ 和X₅ 被獨立地選擇，並且其中：X₁ 為V或I，X₂ 為V或A，X₃ 為F或L，X₄ 為N、D或Y，並且X₅ 為A或T。The preferred optimized anti-ROR1-VH domain amino acid sequence of the invention is a variant of SEQ ID NO: 7 and is represented by SEQ ID NO: 9 (CDR _H residues are indicated by the bottom line): QEQLVESGGG LVQPGGSLRLSCAASGFTFS DYYMS W X ₁ RQA PGKGLEWVAT IYPSSGKTYY ADSX ₂ KG R X ₃ TI SSDNAK X ₄ SLY LQMNSLRAED TAVYYC X ₅ R DS YADDAALFDI WGQGTTVTVS S where: X ₁ , X ₂ , X ₃ , X ₄ and X ₅ are independently selected, and wherein: X ₁ Is V or I, X ₂ is V or A, X ₃ is F or L, X ₄ is N, D or Y, and X ₅ is A or T.

本發明尤其提供這類優化的ROR1-結合分子，其中可變重鏈結構域具有SEQ ID NO:9 的氨基酸序列，其中： (a)X₁ 為V或I，X₂ 為V或A，X₃ 為L，X₄ 為N、D或Y，並且X₅ 為A或T； (b)X₁ 為V或I，X₂ 為V或A，X₃ 為F或L，X₄ 為D或Y，並且X₅ 為A或T； (c)X₁ 為V或I，X₂ 為V或A，X₃ 為F或L，X₄ 為N、D或Y，並且X₅ 為T； (d)X₁ 為V或I，X₂ 為V或A，X₃ 為L，X₄ 為N，並且X₅ 為A； (e)X₁ 為V或I，X₂ 為V或A，X₃ 為F，X₄ 為D，並且X₅ 為A； (f)X₁ 為V或I，X₂ 為V或A，X₃ 為F，X₄ 為N，並且X₅ 為T； (g)X₁ 為V或I，X₂ 為V或A，X₃ 為L，X₄ 為D，並且X₅ 為T； (h)X₁ 為I，X₂ 為A，X₃ 為F或L，X₄ 為N、D或Y，並且 X₅ 為A或T； (i)X₁ 為I，X₂ 為A，X₃ 為F，X₄ 為N，並且X₅ 為A； (j)X₁ 為I，X₂ 為A，X₃ 為L，X₄ 為N，並且X₅ 為A； (k)X₁ 為I，X₂ 為A，X₃ 為F，X₄ 為D，並且X₅ 為A； (l)X₁ 為I，X₂ 為A，X₃ 為F，X₄ 為N，並且X₅ 為T；或 (m)X₁ 為I，X₂ 為A，X₃ 為L，X₄ 為D，並且X₅ 為T。In particular, the invention provides such optimized ROR1-binding molecules wherein the variable heavy domain has the amino acid sequence of SEQ ID NO: 9 , wherein: (a) X ₁ is V or I, X ₂ is V or A, X ₃ is L, X ₄ is N, D or Y, and X ₅ is A or T; (b) X ₁ is V or I, X ₂ is V or A, X ₃ is F or L, and X ₄ is D or Y, and X ₅ is A or T; (c) X ₁ is V or I, X ₂ is V or A, X ₃ is F or L, X ₄ is N, D or Y, and X ₅ is T; d) X ₁ is V or I, X ₂ is V or A, X ₃ is L, X ₄ is N, and X ₅ is A; (e) X ₁ is V or I, X ₂ is V or A, X ₃ is F, X ₄ is D, and X ₅ is A; (f) X ₁ is V or I, X ₂ is V or A, X ₃ is F, X ₄ is N, and X ₅ is T; X ₁ is V or I, X ₂ is V or A, X ₃ is L, X ₄ is D, and X ₅ is T; (h) X ₁ is I, X ₂ is A, and X ₃ is F or L X ₄ is N, D or Y, and X ₅ is A or T; (i) X ₁ is I, X ₂ is A, X ₃ is F, X ₄ is N, and X ₅ is A; (j) X ₁ is I, X ₂ is A, X ₃ is L, X ₄ is N, and X ₅ is A; (k) X ₁ is I, X ₂ is A, X ₃ is F, X ₄ is D, and X ₅ is A; (l) X ₁ is I, X ₂ is A, X ₃ is F, X ₄ is N, and X ₅ T; or (m) X ₁ is I, X ₂ is A, X ₃ is L, X ₄ is D, and X ₅ is T.

在優選的實施方案中，本發明的ROR1-結合分子包括具有SEQ ID NO:9 的氨基酸序列的優化的抗-ROR1-VH結構域，其中： (a)X₁ 為V或I，X₂ 為V或A，X₃ 為L，X₄ 為N、D、或Y，並且X₅ 為A或T； (b)X₁ 為V或I，X₂ 為V或A，X₃ 為F或L，X₄ 為D或Y，並且X₅ 為A或T； (c)X₁ 為V或I，X₂ 為V或A，X₃ 為F或L，X₄ 為N、D、或Y，並且X₅ 為T； (d)X₁ 為V或I，X₂ 為V或A，X₃ 為L，X₄ 為N，並且X₅ 為A； (e)X₁ 為V或I，X₂ 為V或A，X₃ 為F，X₄ 為D，並且X₅ 為A； (f)X₁ 為V或I，X₂ 為V或A，X₃ 為F，X₄ 為N，並且X₅ 為T；或 (g)X₁ 為V或I，X₂ 為V或A，X₃ 為L，X₄ 為D，並且X₅ 為T。In a preferred embodiment, the ROR1-binding molecule of the invention comprises an optimized anti-ROR1-VH domain having the amino acid sequence of SEQ ID NO: 9 , wherein: (a) X ₁ is V or I, X ₂ is V or A, X ₃ is L, X ₄ is N, D, or Y, and X ₅ is A or T; (b) X ₁ is V or I, X ₂ is V or A, X ₃ is F or L X ₄ is D or Y, and X ₅ is A or T; (c) X ₁ is V or I, X ₂ is V or A, X ₃ is F or L, and X ₄ is N, D, or Y, And X ₅ is T; (d) X ₁ is V or I, X ₂ is V or A, X ₃ is L, X ₄ is N, and X ₅ is A; (e) X ₁ is V or I, X ₂ is V or A, X ₃ is F, X ₄ is D, and X ₅ is A; (f) X ₁ is V or I, X ₂ is V or A, X ₃ is F, X ₄ is N, and X ₅ is T; or (g) X ₁ is V or I, X ₂ is V or A, X ₃ is L, X ₄ is D, and X ₅ is T.

在另一個優選的實施方案中，本發明的ROR1-結合分子包括具有SEQ ID NO:9 的氨基酸序列的優化的抗-ROR1-VH結構域，其中X₁ 為I和X₂ 為A，並且其中： (a)X₃ 為F或L，X₄ 為N、D或Y，並且X₅ 為A或T； (b)X₃ 為F，X₄ 為N，且X₅ 為A； (c)X₃ 為L，X₄ 為N，並且X₅ 為A； (d)X₃ 為F，X₄ 為D，並且X₅ 為A； (e)X₃ 為F，X₄ 為N，並且X₅ 為T；或 (f)X₃ 為L，X₄ 為D，並且X₅ 為T。In another preferred embodiment, the ROR1-binding molecule of the invention comprises an optimized anti-ROR1-VH domain having the amino acid sequence of SEQ ID NO: 9 , wherein X ₁ is I and X ₂ is A, and wherein : (a) X ₃ is F or L, X ₄ is N, D or Y, and X ₅ is A or T; (b) X ₃ is F, X ₄ is N, and X ₅ is A; (c) X ₃ is L, X ₄ is N, and X ₅ is A; (d) X ₃ is F, X ₄ is D, and X ₅ is A; (e) X ₃ is F, X ₄ is N, and X ₅ is T; or (f) X ₃ is L, X ₄ is D, and X ₅ is T.

具體地，如本文所提供的，構建並研究了包括親本抗-ROR1-VL結構域(SEQ ID NO:6 )的十四種不同變體的ROR1-結合分子。變異的抗-ROR1-VL結構域被命名為“抗 -ROR1-VL(1) ”、“抗 -ROR1-VL(2) ”、“抗 -ROR1-VL(3) ”、“抗 -ROR1-VL(4) ”、“抗 -ROR1-VL(5) ”、“抗 -ROR1-VL(6) ”、“抗 -ROR1-VL(7) ”、“抗 -ROR1-VL(8) ”、“抗 -ROR1-VL(9) ”、“抗 -ROR1-VL(10) ”、“抗 -ROR1-VL(11) ”、“抗 -ROR1-VL(12) ”、“抗 -ROR1-VL(13) ”和“抗 -ROR1-VL(14) ”。這些變異的VL結構域的氨基酸序列如下示出：Specifically, as provided herein, ROR1-binding molecules comprising fourteen different variants of the parent anti-ROR1-VL domain ( SEQ ID NO: 6 ) were constructed and studied. The variant anti-ROR1-VL domains were named " anti- ROR1-VL(1) ", " anti- ROR1-VL(2) ", " anti- ROR1-VL(3) ", " anti- ROR1- VL(4) ", " anti- ROR1-VL(5) ", " anti- ROR1-VL(6) ", " anti- ROR1-VL(7) ", " anti- ROR1-VL(8) ", " Anti- ROR1-VL(9) ", " Anti- ROR1-VL(10) ", " Anti- ROR1-VL(11) ", " Anti- ROR1-VL(12) ", " Anti- ROR1-VL" (13) "and " anti- ROR1-VL(14) ". The amino acid sequences of these variant VL domains are shown below:

抗-ROR1-VL(1)的氨基酸序列(SEQ ID NO:10 )如下所示(修飾的殘基以底線示)： QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRF - SGS SSGADRYLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLGThe amino acid sequence of anti-ROR1-VL(1) ( SEQ ID NO: 10 ) is shown below (modified residues are shown in the bottom line): QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRF - SGS SSGADRYLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLG

抗-ROR1-VL(2)的氨基酸序列(SEQ ID NO:11 )如下所示(修飾的殘基以底線示)： QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFGSGS SSGAD W YLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLGThe amino acid sequence of anti-ROR1-VL(2) ( SEQ ID NO: 11 ) is shown below (modified residues are shown in the bottom line): QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFGSGS SSGAD W YLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLG

抗-ROR1-VL(3)的氨基酸序列(SEQ ID NO:12 )如下所示(修飾的殘基以底線示)： QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLM N L EGSGSYNKGS GVPDRFGSGS SSGADRYLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLGThe amino acid sequence of anti-ROR1-VL(3) ( SEQ ID NO: 12 ) is shown below (modified residues are shown in the bottom line): QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLM N L EGSGSYNKGS GVPDRFGSGS SSGADRYLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLG

抗-ROR1-VL(4)的氨基酸序列(SEQ ID NO:13 )如下所示(修飾的殘基以底線示)： QLVLTQSPSA SASLG G SVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFGSGS SSGADRYLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLGThe amino acid sequence of anti-ROR1-VL(4) ( SEQ ID NO: 13 ) is shown below (modified residues are shown in the bottom line): QLVLTQSPSA SASLG G SVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFGSGS SSGADRYLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLG

抗-ROR1-VL(5)的氨基酸序列(SEQ ID NO:14 )如下所示(修飾的殘基以底線示)： QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSY S KGS GVPDRFGSGS SSGADRYLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLGThe amino acid sequence of anti-ROR1-VL(5) ( SEQ ID NO: 14 ) is shown below (modified residues are shown in the bottom line): QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSY S KGS GVPDRFGSGS SSGADRYLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLG

抗-ROR1-VL(6)的氨基酸序列(SEQ ID NO:15 )如下所示(修飾的殘基以底線示)： QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFGSG I SSGADRYLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLGThe amino acid sequence of anti-ROR1-VL(6) ( SEQ ID NO: 15 ) is shown below (modified residues are shown in the bottom line): QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFGSG I SSGADRYLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLG

抗-ROR1-VL(7)的氨基酸序列(SEQ ID NO:16 )如下所示(修飾的殘基以底線示)： QLVLTQSPSA SASLGSSV I L TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFGSGS SSGADRYLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLGThe amino acid sequence of anti-ROR1-VL(7) ( SEQ ID NO: 16 ) is shown below (modified residues are shown in the bottom line): QLVLTQSPSA SASLGSSV I L TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFGSGS SSGADRYLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLG

抗-ROR1-VL(8)的氨基酸序列(SEQ ID NO:17 )如下所示(修飾的殘基以底線示)： QLVLTQSPSA SASLGSSV N L TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFGSGS SSGADRYLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLGThe amino acid sequence of anti-ROR1-VL(8) ( SEQ ID NO: 17 ) is shown below (modified residues are shown in the bottom line): QLVLTQSPSA SASLGSSV N L TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFGSGS SSGADRYLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLG

抗-ROR1-VL(9)的氨基酸序列(SEQ ID NO:18 )如下所示(修飾的殘基以底線示)： QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSY T KGS GVPDRFGSGS SSGADRYLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLGThe amino acid sequence of anti-ROR1-VL(9) ( SEQ ID NO: 18 ) is shown below (modified residues are shown in the bottom line): QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSY T KGS GVPDRFGSGS SSGADRYLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLG

抗-ROR1-VL(10)的氨基酸序列(SEQ ID NO:19 )如下所示(修飾的殘基以底線示)： QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFGSGS SSGADRYLTI SSLQSEDEAD YYCGTD N PGN YLFGGGTQLT VLGThe amino acid sequence of anti-ROR1-VL(10) ( SEQ ID NO: 19 ) is shown below (modified residues are shown in the bottom line): QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFGSGS SSGADRYLTI SSLQSEDEAD YYCGTD N PGN YLFGGGTQLT VLG

抗-ROR1-VL(11)的氨基酸序列(SEQ ID NO:20 )如下所示(修飾的殘基以底線示)： QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFGSGS SSGAD W YLTI SSLQSEDEAD YYCGTD N PGN YLFGGGTQLT VLGThe amino acid sequence of anti-ROR1-VL(11) ( SEQ ID NO: 20 ) is shown below (modified residues are shown in the bottom line): QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFGSGS SSGAD W YLTI SSLQSEDEAD YYCGTD N PGN YLFGGGTQLT VLG

抗-ROR1-VL(12)的氨基酸序列(SEQ ID NO:21 )如下所示(修飾的殘基以底線示)： QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFGSG I SSGAD W YLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLGThe amino acid sequence of anti-ROR1-VL (12) ( SEQ ID NO: 21 ) is shown below (modified residues are shown in the bottom line): QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFGSG I SSGAD W YLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLG

抗-ROR1-VL(13)的氨基酸序列(SEQ ID NO:22 )如下所示(修飾的殘基以底線示)： QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFGSG I SSGAD W YLTI SSLQSEDEAD YYCGTD N PGN YLFGGGTQLT VLGThe amino acid sequence of anti-ROR1-VL (13) ( SEQ ID NO: 22 ) is shown below (modified residues are shown in the bottom line): QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFGSG I SSGAD W YLTI SSLQSEDEAD YYCGTD N PGN YLFGGGTQLT VLG

抗-ROR1-VL(14)的氨基酸序列(SEQ ID NO:23 )如下所示(修飾的殘基以底線示)： QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRF - SGS SSGAD W YLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLGThe amino acid sequence of anti-ROR1-VL (14) ( SEQ ID NO: 23 ) is shown below (modified residues are shown in the bottom line): QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRF - SGS SSGAD W YLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLG

所研究的具體修飾概括在表6中，修飾的氨基酸殘基被方框框住，並在圖 7A 中呈現的抗-ROR1-VL的氨基酸序列中用箭頭指示(下面顯示的是Kabat編號)。雖然可以看出，在這些具體的變體抗-ROR1-VL結構域中，多個氨基酸殘基已經被取代或缺失，但當工程化本發明優化的抗-ROR1-VL結構域時不必修飾所有或大部分這些殘基。對於輕鏈可變區域，優選修飾Kabat 位置71(對應於SEQ ID NO:6 的殘基76(X₆ ))處的殘基。尤其地，輕鏈還可以包括在Kabat位置66和92(對應於SEQ ID NO:6 的殘基70(X₅ )和97(X₇ ))中的一個或多個位置處包括修飾。另外，應注意，抗-ROR1-VL在Kabat位置63和64之間包括額外的甘氨酸(G)殘基，因此，輕鏈可進一步包括這樣的額外氨基酸殘基(對應於SEQ ID NO:6 的殘基67(X₄ ))的缺失。在優選的實施方案中，優化的抗-ROR1-VL結構域包括R71W取代，並且可任選地包括：(1)S66I取代和/或(2)Y92N取代，和/或(3)在63和64之間的G殘基的缺失，但是如本文所提供的，可以進行一些其它修飾。本發明還包括這些序列的微小變異，包括例如C-末端和/或N-末端氨基酸殘基的氨基酸取代，其可以被引入以促進亞克隆。The specific modifications studied are summarized in Table 6. The modified amino acid residues are boxed and indicated by arrows in the amino acid sequence of anti-ROR1-VL presented in Figure 7A (Kabat numbering is shown below). Although it can be seen that in these specific variant anti-ROR1-VL domains, multiple amino acid residues have been substituted or deleted, but it is not necessary to modify all of the optimized anti-ROR1-VL domains of the invention. Or most of these residues. For the light chain variable region, preferably the modified Kabat position 71 (corresponding to SEQ ID NO: 766 residues in (X ₆₎₎ at residues. In particular, the light chain may also be included in Kabat position 66 and 92 (corresponding to SEQ ID NO: 706 residues of (X ₅₎ and 97 (X ₇₎₎ at one or more locations include modifications. In addition, it should be noted that the anti-ROR1-VL includes an additional glycine (G) residue between Kabat positions 63 and 64, and thus, the light chain may further comprise such additional amino acid residues (corresponding to SEQ ID NO: 6 Deletion of residue 67 ( X ₄ )). In a preferred embodiment, the optimized anti-ROR1-VL domain comprises an R71W substitution, and may optionally include: (1) S66I substitution and/or (2) Y92N substitution, and/or (3) at 63 and Deletion of the G residue between 64, but as provided herein, some other modifications can be made. The invention also includes minor variations of these sequences, including, for example, amino acid substitutions of C-terminal and/or N-terminal amino acid residues, which can be introduced to facilitate subcloning.

在各種實施方案中，本發明的ROR1-結合分子包括優化的抗-ROR1-VL結構域，所述VL結構域優選地包括選自SEQ ID NO:11 、19 、20 、21 、22 和23 的氨基酸序列。在優選的實施方案中，本發明的ROR1-結合分子包括優化的抗-ROR1-VH結構域，其包括SEQ ID NO:11 或SEQ ID NO:23 的氨基酸序列。In various embodiments, an ROR1-binding molecule of the invention comprises an optimized anti-ROR1-VL domain, preferably comprising a SEQ ID NO: 11 , 19 , 20 , 21 , 22 and 23 Amino acid sequence . In a preferred embodiment, the ROR1-binding molecule of the invention comprises an optimized anti-ROR1-VH domain comprising the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 23 .

尤其地，如本文所提供的，構建並研究了包括親本抗-ROR1-VH結構域(SEQ ID NO:7 )的八種不同變體的ROR1-結合分子。變體抗-ROR1-VH結構域被命名為“抗 -ROR1-VH(1) ”、“抗 -ROR1-VH(2) ”、“抗 -ROR1-VH(3) ”，“抗 -ROR1-VH(4) ”、“抗 -ROR1-VH(5) ”、“抗 -ROR1-VH(6) ”、“抗 -ROR1-VH(7) ”和“抗 -ROR1-VH(8) ”。還提供了可以被構建的另外的變體(命名為“抗 -ROR1-VH(9) ”)。這些變異的VH結構域的氨基酸序列如下所示：In particular, as provided herein, ROR1-binding molecules comprising eight different variants of the parent anti-ROR1-VH domain ( SEQ ID NO: 7 ) were constructed and studied. The variant anti-ROR1-VH domain was named " anti- ROR1-VH(1) ", " anti- ROR1-VH(2) ", " anti- ROR1-VH(3) ", " anti- ROR1- VH(4) ", " anti- ROR1-VH(5) ", " anti- ROR1-VH(6) ", " anti- ROR1-VH(7) " and " anti- ROR1-VH(8) ". Additional variants (designated " anti- ROR1-VH(9) ")) that can be constructed are also provided. The amino acid sequences of these variant VH domains are as follows:

抗-ROR1-VH(1)的氨基酸序列(SEQ ID NO:24 )如下所示(修飾的殘基以底線示)： QEQLVESGGG LVQPGGSLRL SCAASGFTFS DYYMSWVRQA PGKGLEWVAT IYPSSGKTYY ADSVKGR L TI SSDNAKNSLY LQMNSLRAED TAVYYCARDS YADDAALFDI WGQGTTVTVS SThe amino acid sequence of anti-ROR1-VH(1) ( SEQ ID NO: 24 ) is shown below (modified residues are shown in the bottom line): QEQLVESGGG LVQPGGSLRL SCAASGFTFS DYYMSWVRQA PGKGLEWVAT IYPSSGKTYY ADSVKGR L TI SSDNAKNSLY LQMNSLRAED TAVYYCARDS YADDAALFDI WGQGTTVTVS S

抗-ROR1-VH(2)的氨基酸序列(SEQ ID NO:25 )如下所示(修飾的殘基以底線示)： QEQLVESGGG LVQPGGSLRL SCAASGFTFS DYYMSWVRQA PGKGLEWVAT IYPSSGKTYY ADSVKGRFTI SSDNAK D SLY LQMNSLRAED TAVYYCARDS YADDAALFDI WGQGTTVTVS SThe amino acid sequence of anti-ROR1-VH(2) ( SEQ ID NO: 25 ) is shown below (modified residues are shown in the bottom line): QEQLVESGGG LVQPGGSLRL SCAASGFTFS DYYMSWVRQA PGKGLEWVAT IYPSSGKTYY ADSVKGRFTI SSDNAK D SLY LQMNSLRAED TAVYYCARDS YADDAALFDI WGQGTTVTVS S

抗-ROR1-VH(3)的氨基酸序列(SEQ ID NO:26 )如下所示(修飾的殘基以底線示)： QEQLVESGGG LVQPGGSLRL SCAASGFTFS DYYMSWVRQA PGKGLEWVAT IYPSSGKTYY ADSVKGRFTI SSDNAKNSLY LQMNSLRAED TAVYYC T RDS YADDAALFDI WGQGTTVTVS SThe amino acid sequence of anti-ROR1-VH(3) ( SEQ ID NO: 26 ) is shown below (modified residues are shown in the bottom line): QEQLVESGGG LVQPGGSLRL SCAASGFTFS DYYMSWVRQA PGKGLEWVAT IYPSSGKTYY ADSVKGRFTI SSDNAKNSLY LQMNSLRAED TAVYYC T RDS YADDAALFDI WGQGTTVTVS S

抗-ROR1-VH(4)的氨基酸序列(SEQ ID NO:27 )如下所示(修飾的殘基以底線示)： QEQLVESGGG LVQPGGSLRL SCAASGFTFS DYYMSWVRQA PGKGLEWVAT IYPSSGKTYY ADSVKGRFTI SSDNAK Y SLY LQMNSLRAED TAVYYCARDS YADDAALFDI WGQGTTVTVS SThe amino acid sequence of anti-ROR1-VH(4) ( SEQ ID NO:27 ) is shown below (modified residues are shown in the bottom line): QEQLVESGGG LVQPGGSLRL SCAASGFTFS DYYMSWVRQA PGKGLEWVAT IYPSSGKTYY ADSVKGRFTI SSDNAK Y SLY LQMNSLRAED TAVYYCARDS YADDAALFDI WGQGTTVTVS S

抗-ROR1-VH(5)的氨基酸序列(SEQ ID NO:28 )如下所示(修飾的殘基以底線示)： QEQLVESGGG LVQPGGSLRL SCAASGFTFS DYYMSWVRQA PGKGLEWVAT IYPSSGKTYY ADSVKGRFTI SSDNAKNSLY LQMNSLRAED TAVYYCARDS YADDAALF A I WGQGTTVTVS SThe amino acid sequence of anti-ROR1-VH(5) ( SEQ ID NO: 28 ) is shown below (modified residues are shown in the bottom line): QEQLVESGGG LVQPGGSLRL SCAASGFTFS DYYMSWVRQA PGKGLEWVAT IYPSSGKTYY ADSVKGRFTI SSDNAKNSLY LQMNSLRAED TAVYYCARDS YADDAALF A I WGQGTTVTVS S

抗-ROR1-VH(6)的氨基酸序列(SEQ ID NO:29 )如下所示(修飾的殘基以底線示)： QEQLVESGGG LVQPGGSLRL SCAASGFTFS DYYMSWVRQA PGKGLEWVAT IYPSSGKTYY ADSVKGRFTI SSDNAKNSLY LQMNSLRAED TAVYYCARDS YADDAALF Y I WGQGTTVTVS SThe amino acid sequence of anti-ROR1-VH(6) ( SEQ ID NO: 29 ) is shown below (modified residues are shown in the bottom line): QEQLVESGGG LVQPGGSLRL SCAASGFTFS DYYMSWVRQA PGKGLEWVAT IYPSSGKTYY ADSVKGRFTI SSDNAKNSLY LQMNSLRAED TAVYYCARDS YADDAALF Y I WGQGTTVTVS S

抗-ROR1-VH(7)的氨基酸序列(SEQ ID NO:30 )如下所示(修飾的殘基以底線示)： QEQLVESGGG LVQPGGSLRL SCAASGFTFS DYYMSWVRQA PGKGLEWVAT IYPSSGKTYY ADSVKGR L TI SSDNAK D SLY LQMNSLRAED TAVYYC T RDS YADDAALFDI WGQGTTVTVS SThe amino acid sequence of anti-ROR1-VH(7) ( SEQ ID NO: 30 ) is shown below (modified residues are shown in the bottom line): QEQLVESGGG LVQPGGSLRL SCAASGFTFS DYYMSWVRQA PGKGLEWVAT IYPSSGKTYY ADSVKGR L TI SSDNAK D SLY LQMNSLRAED TAVYYC T RDS YADDAALFDI WGQGTTVTVS S

抗-ROR1-VH(8)的氨基酸序列(SEQ ID NO:31 )如下所示(修飾的殘基以底線示)： QEQLVESGGG LVQPGGSLRL SCAASGFTFS DYYMSW I RQA PGKGLEWVAT IYPSSGKTYY ADS A KGR L TI SSDNAK D SLY LQMNSLRAED TAVYYC T RDS YADDAALFDI WGQGTTVTVS SThe amino acid sequence of anti-ROR1-VH(8) ( SEQ ID NO: 31 ) is shown below (modified residues are shown in the bottom line): QEQLVESGGG LVQPGGSLRL SCAASGFTFS DYYMSW I RQA PGKGLEWVAT IYPSSGKTYY ADS A KGR L TI SSDNAK D SLY LQMNSLRAED TAVYYC T RDS YADDAALFDI WGQGTTVTVS S

抗-ROR1-VH(9)的氨基酸序列(SEQ ID NO:32 )如下所示(修飾的殘基以底線示)： QEQLVESGGG LVQPGGSLRL SCAASGFTFS DYYMSW I RQA PGKGLEWVAT IYPSSGKTYY ADS A KGRFTI SSDNAKNSLY LQMNSLRAED TAVYYCARDS YADDAALFDI WGQGTTVTVS SThe amino acid sequence of anti-ROR1-VH(9) ( SEQ ID NO: 32 ) is shown below (modified residues are shown in the bottom line): QEQLVESGGG LVQPGGSLRL SCAASGFTFS DYYMSW I RQA PGKGLEWVAT IYPSSGKTYY ADS A KGRFTI SSDNAKNSLY LQMNSLRAED TAVYYCARDS YADDAALFDI WGQGTTVTVS S

所研究的具體修飾概括在表6中，修飾的氨基酸殘基被方框框住，並在圖 7B 中呈現的抗-ROR1-VH的氨基酸序列中用箭頭指示(下面顯示的是Kabat編號)。雖然可以看出，在這些具體的優化的抗-ROR1-VH結構域中多個氨基酸殘基已經被取代或缺失，但當工程化本發明優化的抗-ROR1-VH結構域時不必修飾所有或大部分這些殘基。對於重鏈可變區域，優選修飾Kabat 位置67、76和93(對應於SEQ ID NO:9 的殘基68 (X₃ )、77 (X₄ )和97 (X₅ ))中的一個或多個殘基。另外或者可選地，重鏈可以在Kabat位置37和67(對應於SEQ ID NO:9 的殘基37 (X₁ )和64 (X₂ ))中的一個或多個位置處包括修飾。在優選的實施方案中，優化的抗-ROR1-VH結構域包括：(1)F67L取代和/或(2)N76D取代，和/或(3)A93T取代，和/或(4)V37I取代，和/或(5)V63A取代，但是如本文所提供的，可以進行一些其它修飾。本發明還包括這些序列的微小變異，包括例如C-末端和/或N-末端氨基酸殘基的氨基酸取代，其可以被引入以促進亞克隆。The specific modifications studied are summarized in Table 6. The modified amino acid residues are boxed and indicated by arrows in the amino acid sequence of anti-ROR1-VH presented in Figure 7B (Kabat numbering is shown below). Although it can be seen that multiple amino acid residues have been substituted or deleted in these specific optimized anti-ROR1-VH domains, it is not necessary to modify all or when engineering the optimized anti-ROR1-VH domain of the invention. Most of these residues. For the heavy chain variable region, preferably the modified Kabat positions 67,76 and 93 (corresponding to SEQ ID NO: 9 residues _{68 (X 3), 77 (} X 4) and 97 ₍₅₎ X) or in a Residues. Additionally or alternatively, the heavy chain can include modifications at one or more positions in Kabat positions 37 and 67 (corresponding to residues 37 ( X ₁ ) and 64 ( X ₂ ) of SEQ ID NO: 9 ). In a preferred embodiment, the optimized anti-ROR1-VH domain comprises: (1) F67L substitution and/or (2) N76D substitution, and/or (3) A93T substitution, and/or (4) V37I substitution, And/or (5) V63A is substituted, but as provided herein, some other modifications can be made. The invention also includes minor variations of these sequences, including, for example, amino acid substitutions of C-terminal and/or N-terminal amino acid residues, which can be introduced to facilitate subcloning.

在各種實施方案中，本發明的ROR1-結合分子包括優化的抗-ROR1-VH結構域，所述VH結構域優選地包括選自SEQ ID NO:24 、25 、26 、27 、30 、31 和32 的氨基酸序列。在優選的實施方案中，本發明的ROR1-結合分子包括優化的抗-ROR1-VH結構域，其包括SEQ ID NO: 26 、30 、31 或32 的氨基酸序列。In various embodiments, the ROR1-binding molecules of the invention comprise an optimized anti-ROR1-VH domain, preferably comprising a SEQ ID NO: 24 , 25 , 26 , 27 , 30 , 31 and Amino acid sequence of 32 . In a preferred embodiment, the ROR1-binding molecule of the invention comprises an optimized anti-ROR1-VH domain comprising the amino acid sequence of SEQ ID NO: 26 , 30 , 31 or 32 .

在其它實施方案中，本發明的ROR1-結合分子包括優化的抗-ROR1-VL結構域，並且還包括優化的抗-ROR1-VH結構域。本發明的ROR1-結合分子可以包括本文所述的優化的抗-ROR1-VL和抗-ROR1-VH結構域的任何組合： In other embodiments, the ROR1-binding molecules of the invention include an optimized anti-ROR1-VL domain, and also include an optimized anti-ROR1-VH domain. The ROR1-binding molecules of the invention can include any combination of the optimized anti-ROR1-VL and anti-ROR1-VH domains described herein:

在各種實施方案中，本發明的ROR1-結合分子包括以下組合中的一個： In various embodiments, the ROR1-binding molecules of the invention comprise one of the following combinations:

尤其優選的組合是： A particularly preferred combination is:

本發明特別包括ROR1-結合分子，其包括：(i)如上所提供的優化的抗-ROR1-VL和/或VH結構域，和(ii)Fc區域。在具體實施方案中，本發明的ROR1-結合分子是單克隆抗體，其包括：(i)如上所提供的優化的抗-ROR1-VL和/或VH結構域，和(ii)Fc區域。在其它實施方案中，本發明的ROR1-結合分子選自：單克隆抗體、多特異性抗體、合成抗體、嵌合抗體、單鏈Fv(scFv)、單鏈抗體、Fab片段、F(ab')片段、二硫鍵連接的雙特異性Fvs(sdFv)、BiTEs、雙抗體和三價結合分子。The invention specifically includes ROR1-binding molecules comprising: (i) an optimized anti-ROR1-VL and/or VH domain as provided above, and (ii) an Fc region. In a specific embodiment, the ROR1-binding molecule of the invention is a monoclonal antibody comprising: (i) an optimized anti-ROR1-VL and/or VH domain as provided above, and (ii) an Fc region. In other embodiments, the ROR1-binding molecules of the invention are selected from the group consisting of: monoclonal antibodies, multispecific antibodies, synthetic antibodies, chimeric antibodies, single chain Fv (scFv), single chain antibodies, Fab fragments, F (ab' a fragment, a disulfide-linked bispecific Fvs (sdFv), a BiTEs, a diabody, and a trivalent binding molecule.

V.V. 嵌合抗原受體Chimeric antigen receptor

本發明的ROR1-結合分子可以是單特異性單鏈分子，例如單鏈可變片段(“抗 -ROR1-scFv ”)或嵌合抗原受體(“抗 -ROR1-CAR ”)。如上所述，scFv通過經由短連接肽將輕鏈和重鏈可變結構域連接在一起來製備。第一代CAR通常具有來自CD3ζ鏈的細胞內結構域，其是來自內源性TCR的信號的主要傳遞者。第二代CAR具有從各種共刺激蛋白受體(例如CD28、41BB、ICOS等)到CAR的胞質尾區的另外的細胞內信號傳導結構域，以向T細胞提供另外的信號。第三代CAR組合多種信號傳導結構域，例如CD3z-CD28-41BB或CD3z-CD28-OX40，以進一步增加效力(Tettamanti, S.等(2013) “Targeting Of Acute Myeloid Leukaemia By Cytokine-Induced Killer Cells Redirected With A Novel CD123-Specific Chimeric AntigenReceptor ,” Br. J. Haematol. 161:389-401；Gill, S.等(2014) “Efficacy Against Human Acute Myeloid Leukemia And Myeloablation Of Normal Hematopoiesis In A Mouse Model Using Chimeric Antigen Receptor-Modified T Cells ,” Blood 123(15): 2343-2354；Mardiros, A.等(2013) “T Cells Expressing CD123-Specific Chimeric Antigen Receptors Exhibit Specific Cytolytic Effector Functions And Antitumor Effects Against Human Acute Myeloid Leukemia ,” Blood 122:3138-3148；Pizzitola, I.等(2014) “Chimeric Antigen Receptors Against CD33/CD123 Antigens Efficiently Target Primary Acute Myeloid Leukemia Cells in vivo ,” Leukemia doi:10.1038/leu.2014.62)。The ROR1-binding molecule of the invention may be a monospecific single chain molecule, such as a single chain variable fragment (" anti- ROR1-scFv ") or a chimeric antigen receptor (" anti- ROR1-CAR "). As described above, scFv is prepared by linking the light chain and heavy chain variable domains together via a short linker peptide. First generation CARs typically have an intracellular domain from the CD3 ζ chain, which is the primary transmitter of signals from endogenous TCRs. Second generation CARs have additional intracellular signaling domains from various costimulatory protein receptors (eg, CD28, 41BB, ICOS, etc.) to the cytoplasmic tail of CAR to provide additional signals to T cells. The third generation of CAR combines multiple signaling domains, such as CD3z-CD28-41BB or CD3z-CD28-OX40, to further increase potency (Tettamanti, S. et al. (2013) " Targeting Of Acute Myeloid Leukaemia By Cytokine-Induced Killer Cells Redirected With A Novel CD123-Specific Chimeric Antigen Receptor ," Br. J. Haematol. 161:389-401; Gill, S. et al. (2014) " Efficacy Against Human Acute Myeloid Leukemia And Myeloablation Of Normal Hematopoiesis In A Mouse Model Using Chimeric Antigen Receptor-Modified T Cells ,” Blood 123(15): 2343-2354; Mardiros, A. et al. (2013) “ T Cells Expressing CD123-Specific Chimeric Antigen Receptors Exhibit Specific Cytolytic Effector Functions And Antitumor Effects Against Human Acute Myeloid Leukemia ,” Blood 122: 3138-3148; Pizzitola, I. et al. (2014) " Chimeric Antigen Receptors Against CD33/CD123 Antigens Efficiently Target Primary Acute Myeloid Leukemia Cells in vivo ," Leukemia doi: 10.1038/leu. 2014.62).

本發明的抗-ROR1-CAR包括與受體的細胞內結構域融合的抗-ROR1-scFv。抗-ROR1-scFv的可變輕鏈和可變重鏈結構域選自本文公開的任何優化的抗-ROR1-VL結構域和抗-ROR1-VH結構域。優選地，VL結構域選自：抗-ROR1-VL(2)(SEQ ID NO:11 )、抗-ROR1-VL(11)(SEQ ID NO:20 )、抗-ROR1-VL (12)(SEQ ID NO:21 )、抗-ROR1-VL(13)(SEQ ID NO:22 )和抗-ROR1-VL(14)(SEQ ID NO:23 )。優選地，VH域選自：抗-ROR1-VH(3)(SEQ ID NO:26 )、抗-ROR1-VH(7)(SEQ ID NO:30 )、抗-ROR1-VH (8)(SEQ ID NO:31 )和抗-ROR1-VH(9)(SEQ ID NO:32 )。因此，對於這類抗-ROR1-CAR的這類抗-ROR1-scFv，優化的抗-ROR1-VL和抗-ROR1-VH結構域的以下組合是優選的： The anti-ROR1-CAR of the invention comprises an anti-ROR1-scFv fused to the intracellular domain of the receptor. The variable light and variable heavy chain domains of the anti-ROR1-scFv are selected from any of the optimized anti-ROR1-VL domains and anti-ROR1-VH domains disclosed herein. Preferably, the VL domain is selected from the group consisting of: anti-ROR1-VL(2) ( SEQ ID NO: 11 ), anti-ROR1-VL (11) ( SEQ ID NO: 20 ), anti-ROR1-VL (12) ( SEQ ID NO: 21 ), anti-ROR1-VL (13) ( SEQ ID NO: 22 ) and anti-ROR1-VL (14) ( SEQ ID NO: 23 ). Preferably, the VH domain is selected from the group consisting of: anti-ROR1-VH(3) ( SEQ ID NO:26 ), anti-ROR1-VH(7) ( SEQ ID NO:30 ), anti-ROR1-VH (8) ( SEQ ID NO: 31 ) and anti-ROR1-VH (9) ( SEQ ID NO: 32 ). Thus, for such anti-ROR1-scFvs of such anti-ROR1-CAR, the following combinations of optimized anti-ROR1-VL and anti-ROR1-VH domains are preferred:

本發明的抗-ROR1-CAR的細胞內結構域優選地選自以下任一種的細胞內結構域：41BB-CD3ζ、b2c-CD3ζ、CD28、CD28-4-1BB-CD3ζ、CD28-CD3ζ、 CD28-FcεRIγ、CD28mut-CD3ζ、CD28-OX40-CD3ζ、CD28-OX40-CD3ζ、CD3ζ、CD4-CD3ζ、CD4-FcεRIγ、CD8-CD3ζ、FcεRIγ、FcεRIγCAIX、Heregulin-CD3ζ、IL-13- CD3ζ或Ly49H-CD3ζ (Tettamanti, S.等(2013) “Targeting Of Acute Myeloid Leukaemia By Cytokine-Induced Killer Cells Redirected With A Novel CD123-Specific Chimeric AntigenReceptor ,” Br. J. Haematol. 161:389-401；Gill, S.等(2014) “Efficacy Against Human Acute Myeloid Leukemia And Myeloablation Of Normal Hematopoiesis In A Mouse Model Using Chimeric Antigen Receptor-Modified T Cells ,” Blood 123(15): 2343-2354；Mardiros, A.等(2013) “T Cells Expressing CD123-Specific Chimeric Antigen Receptors Exhibit Specific Cytolytic Effector Functions And Antitumor Effects Against Human Acute Myeloid Leukemia ,” Blood 122:3138-3148；Pizzitola, I.等(2014) “Chimeric Antigen Receptors Against CD33/CD123 Antigens Efficiently Target Primary Acute Myeloid Leukemia Cells in vivo ,” Leukemia doi:10.1038/leu.2014.62)。The intracellular domain of the anti-ROR1-CAR of the present invention is preferably selected from the intracellular domain of any of the following: 41BB-CD3ζ, b2c-CD3ζ, CD28, CD28-4-1BB-CD3ζ, CD28-CD3ζ, CD28- FcεRIγ, CD28mut-CD3ζ, CD28-OX40-CD3ζ, CD28-OX40-CD3ζ, CD3ζ, CD4-CD3ζ, CD4-FcεRIγ, CD8-CD3ζ, FcεRIγ, FcεRIγCAIX, Heregul-CD3ζ, IL-13-CD3ζ or Ly49H-CD3ζ ( Tettamanti, S. et al. (2013) “ Targeting Of Acute Myeloid Leukaemia By Cytokine-Induced Killer Cells Redirected With A Novel CD123-Specific Chimeric Antigen Receptor ,” Br. J. Haematol. 161:389-401; Gill, S. et al. 2014) “ Efficacy Against Human Acute Myeloid Leukemia And Myeloablation Of Normal Hematopoiesis In A Mouse Model Using Chimeric Antigen Receptor-Modified T Cells ,” Blood 123(15): 2343-2354; Mardiros, A. et al. (2013) “ T Cells Expressing CD123-Specific Chimeric Antigen Receptors Exhibit Specific Cytolytic Effector Functions And Antitumor Effects Against Human Acute Myeloid Leukemia ," Blood 122: 3138-3148; Pizzitola, I. et al. (2014) " Chimeric Antigen Receptors Against CD33/CD123 Antigens Efficiently Target Primary Acute Myeloid Leukemia Cells in vivo ," Leukemia doi: 10.1038/leu. 2014. 62).

VI.VI. 多特異性Multispecific ROR1-ROR1- 結合分子Binding molecule

本發明還涉及這樣的ROR1結合分子：其包括表位-結合位點(優選地包括本發明的優化的抗-ROR1-VL結構域和/或本發明的優化的抗-ROR1-VH結構域)，並且還包括免疫特異性結合第二表位的第二表位-結合位點，其中這樣的第二表位是(i)ROR1的不同表位，或(ii)不是ROR1的分子的表位。這類三特異性或多特異性ROR1-結合分子優選地包括識別對靶細胞或組織類型獨特的一組抗原的表位-結合位元點的組合。尤其地，本發明涉及能夠結合ROR1的表位和存在於效應細胞(特別是T淋巴細胞、自然殺傷(NK)細胞或其他單核細胞)表面上的分子的表位的三特異性或多特異性ROR1結合分子。例如，本發明的這類ROR1-結合分子可以被構建為包括免疫特異性結合CD2、CD3、CD8、CD16、T細胞受體(TCR)或NKG2D的表位結合位點。The invention also relates to a ROR1 binding molecule comprising an epitope-binding site (preferably comprising an optimized anti-ROR1-VL domain of the invention and/or an optimized anti-ROR1-VH domain of the invention) And further comprising a second epitope-binding site that immunospecifically binds to a second epitope, wherein such a second epitope is (i) a different epitope of ROR1, or (ii) an epitope of a molecule that is not ROR1 . Such trispecific or multispecific ROR1-binding molecules preferably include a combination of epitope-binding bit points that recognize a set of antigens that are unique to the target cell or tissue type. In particular, the present invention relates to a trispecific or multispecific epitope capable of binding to an epitope of ROR1 and an epitope present on the surface of an effector cell, particularly a T lymphocyte, natural killer (NK) cell or other monocyte. A sexual ROR1 binding molecule. For example, such a ROR1-binding molecule of the invention can be constructed to include an epitope binding site that immunospecifically binds to CD2, CD3, CD8, CD16, T cell receptor (TCR) or NKG2D.

本發明的一個實施方案涉及能夠結合“第一表位”和“第二表位”的雙特異性ROR1-結合分子，這樣的表位彼此不相同。這類雙特異性分子包括能夠結合第一表位的“VL1 ”/“VH1 ”結構域和能夠結合第二表位的“VL2 ”/“VH2 ”結構域。符號“VL1 ”和“VH1 ”分別表示結合這類雙特異性分子的“第一”表位元的可變輕鏈結構域和可變重鏈結構域。類似地，符號“VL2 ”和“VH2 ”分別表示結合這類雙特異性分子的“第二”表位元的輕鏈可變結構域和重鏈可變結構域。將具體表位命名為第一表位還是第二表位是不重要的；這類符號僅與本發明的結合分子的多肽鏈的結構域的存在和取向有關。在一個實施方案中，這樣的表位之一是人ROR1的表位，另一個是ROR1的不同表位，或不是ROR1的分子的表位。在具體的實施方案中，這樣的表位之一是人ROR1的表位，另一個是存在於效應細胞，例如T淋巴細胞、自然殺傷(NK)細胞或其他單核細胞，的表面上的分子(例如，CD2、CD3、CD8、CD16、T-細胞受體(TCR)、NKG2D等)的表位。在某些實施方案中，雙特異性分子包括多於兩個的表位-結合位點。這樣的雙特異性分子將結合ROR1的至少一個表位和不是ROR1的分子的至少一個表位，並且可以進一步結合ROR1的另外的表位和/或不是ROR1的分子的另外的表位。One embodiment of the invention relates to a bispecific ROR1-binding molecule capable of binding a "first epitope" and a "second epitope", such epitopes being different from each other. Such bispecific molecules include the " VL1 "/" VH1 " domain capable of binding to the first epitope and the " VL2 "/" VH2 " domain capable of binding to the second epitope. The symbols " VL1 " and " VH1 " respectively denote the variable light chain domain and variable heavy chain domain that bind to the "first" epitope of such bispecific molecules. Similarly, the symbols " VL2 " and " VH2 " denote the light chain variable domain and the heavy chain variable domain, respectively, which bind to the "second" epitope of such bispecific molecules. It is not important to name a particular epitope as a first epitope or a second epitope; such symbols are only related to the presence and orientation of the domains of the polypeptide chains of the binding molecules of the invention. In one embodiment, one of such epitopes is an epitope of human ROR1, the other is a different epitope of ROR1, or an epitope of a molecule other than ROR1. In a specific embodiment, one of such epitopes is an epitope of human ROR1 and the other is a molecule present on the surface of an effector cell, such as a T lymphocyte, natural killer (NK) cell or other monocyte. Epitopes (eg, CD2, CD3, CD8, CD16, T-cell receptor (TCR), NKG2D, etc.). In certain embodiments, the bispecific molecule comprises more than two epitope-binding sites. Such a bispecific molecule will bind to at least one epitope of ROR1 and at least one epitope of a molecule that is not ROR1, and may further bind to an additional epitope of ROR1 and/or an additional epitope of a molecule that is not ROR1.

本發明尤其涉及雙特異性、三特異性和多特異性ROR1-結合分子(例如雙特異性抗體、雙特異性雙抗體、三價結合分子等)，其具有抗體的表位-結合片段(例如VL和VH結構域)，使它們能夠協調地結合ROR1的至少一個表位和不是ROR1的第二分子的至少一個表位。這些分子的多肽結構域的VL和VH結構域的選擇被協調，以便構成這類多特異性ROR1-結合分子的多肽鏈組裝的多肽鏈形成對至少一個ROR1的表位特異性的至少一個功能性表位-結合位點和對不是ROR1的分子的至少一個表位特異性的至少一個功能性表位-結合位點。優選地，雙特異性ROR1-結合分子包括本文提供的優化的抗-ROR1-VL和/或VH結構域。In particular, the present invention relates to bispecific, trispecific and multispecific ROR1-binding molecules (eg, bispecific antibodies, bispecific diabodies, trivalent binding molecules, etc.) having epitope-binding fragments of antibodies (eg, VL and VH domains) enable them to coordinately bind at least one epitope of ROR1 and at least one epitope of a second molecule that is not ROR1. The selection of the VL and VH domains of the polypeptide domains of these molecules is coordinated such that the polypeptide chains assembled into the polypeptide chains of such multispecific ROR1-binding molecules form at least one functionality specific for the epitope specificity of at least one ROR1 An epitope-binding site and at least one functional epitope-binding site specific for at least one epitope of a molecule that is not ROR1. Preferably, the bispecific ROR1-binding molecule comprises the optimized anti-ROR1-VL and/or VH domains provided herein.

A.A. 雙特異性抗體Bispecific antibody

本發明包括能夠同時結合ROR1的表位和不是ROR1的分子的表位的雙特異性抗體。在一些實施方案中，能夠同時結合ROR1和不是ROR1的第二分子的雙特異性抗體是使用以下文獻中描述的任何方法製備的：PCT公開號WO 1998/002463、WO 2005/070966、WO 2006/107786 、WO 2007/024715、WO 2007/075270、WO 2006/107617、WO 2007/046893、WO 2007/146968、WO 2008/003103、WO 2008/003116、WO 2008/027236、WO 2008/024188、WO 2009/132876、WO 2009/018386、WO 2010/028797、WO2010028796、WO 2010/028795、WO 2010/108127、WO 2010/136172、WO 2011/086091、WO 2011/133886、WO 2012/009544、WO 2013/003652、WO 2013/070565、WO 2012/162583、WO 2012/156430、WO 2013/174873和WO 2014/022540，其各自通過引用以其整體併入本文。The invention encompasses bispecific antibodies that are capable of binding both an epitope of ROR1 and an epitope of a molecule that is not ROR1. In some embodiments, a bispecific antibody capable of binding both ROR1 and a second molecule that is not ROR1 is prepared using any of the methods described in PCT Publication No. WO 1998/002463, WO 2005/070966, WO 2006/ 107786, WO 2007/024715, WO 2007/075270, WO 2006/107617, WO 2007/046893, WO 2007/146968, WO 2008/003103, WO 2008/003116, WO 2008/027236, WO 2008/024188, WO 2009/ 132876, WO 2009/018386, WO 2010/028797, WO 2010028796, WO 2010/028795, WO 2010/108127, WO 2010/136172, WO 2011/086091, WO 2011/133886, WO 2012/009544, WO 2013/003652, WO 2013/070565, WO 2012/162583, WO 2012/156430, WO 2013/174873, and WO 2014/022540, each of which is incorporated herein in entirety by reference.

B.B. 缺少lack FcFc 區域的雙特異性雙抗體Regional bispecific diabodies

本發明的一個實施方案涉及能夠結合第一表位和第二表位的雙特異性雙抗體，其中第一表位是人ROR1的表位，第二表位是不是ROR1的分子的表位，所述不是ROR1的分子優選是存在於效應細胞例如T淋巴細胞、自然殺傷(NK)細胞或其他單核細胞表面上的分子(例如CD2、CD3、CD8、CD16、T-細胞受體(TCR)、NKG2D等)。這樣的雙抗體包括第一多肽鏈和第二多肽鏈，並且最優選地由第一多肽鏈和第二多肽鏈組成，其序列允許多肽鏈彼此共價結合以形成共價締合的雙抗體，其能夠同時結合ROR1的表位和第二表位。One embodiment of the invention relates to a bispecific diabodies capable of binding a first epitope and a second epitope, wherein the first epitope is an epitope of human ROR1 and the second epitope is an epitope of a molecule that is not ROR1, The molecule that is not ROR1 is preferably a molecule that is present on the surface of effector cells such as T lymphocytes, natural killer (NK) cells, or other monocytes (eg, CD2, CD3, CD8, CD16, T-cell receptor (TCR). , NKG2D, etc.). Such diabody includes a first polypeptide chain and a second polypeptide chain, and most preferably consists of a first polypeptide chain and a second polypeptide chain, the sequence of which allows the polypeptide chains to covalently bond to each other to form a covalent association A diabody that is capable of binding both the epitope of ROR1 and the second epitope.

雙特異性雙抗體的這類實施方案的第一多肽鏈在N-末端至C-末端方向包括：N-末端、能夠結合第一或第二表位的單克隆抗體的VL結構域(即VL_抗 _-ROR1-VL 或VL_表位 ₂ )、第一間插間隔體肽(連接體1)、能夠結合第二表位(如果這樣的第一多肽鏈含有VL_抗 _{-ROR1- VL} )或ROR1(如果這樣的第一多肽鏈含有VL_表位 ₂ )的單克隆抗體的VH結構域、任選地含有半胱氨酸殘基的第二間插間隔體肽(連接體2)、異源二聚體-促進結構域和C-末端(圖 1 )。The first polypeptide chain of such embodiments of the bispecific diabody comprises, in the N-terminal to C-terminal direction: the VL domain of the N-terminal, monoclonal antibody capable of binding the first or second epitope (ie VL _anti- _ROR1-VL or VL _epitope ₂ ), first intervening spacer peptide (linker 1), capable of binding to a second epitope (if such a first polypeptide chain contains VL _anti- _ROR1-VL ) or a VH domain of a monoclonal antibody of ROR1 (if such a first polypeptide chain contains VL _epitope ₂ ), a second intervening spacer peptide (linker 2) optionally containing a cysteine residue, Source dimer-promoting domain and C-terminus ( Figure 1 ).

雙特異性雙抗體的該實施方案的第二多肽鏈在N-末端至C-末端方向包括：N-末端、能夠結合第一或第二表位的單克隆抗體的VL結構域(即，VL_抗 _-ROR1-VL 或VL_表位 ₂ ，並且是未被選擇包括在雙抗體的第一多肽鏈中的VL結構域)、間插間隔體肽(連接體1)、能夠結合第二表位(如果這樣的第二多肽鏈含有VL_抗 _-ROR1-VL )或結合ROR1(如果這樣的第二多肽鏈含有VL_表位 ₂ )的單克隆抗體的VH結構域、任選地含有半胱氨酸殘基的第二間插間隔體肽(連接體2)、異源二聚體-促進結構域和C-末端(圖 1 )。The second polypeptide chain of this embodiment of the bispecific diabody comprises, in the N-terminal to C-terminal direction: an N-terminal, VL domain of a monoclonal antibody capable of binding to the first or second epitope (ie, VL _anti- _ROR1-VL or VL _epitope ₂ , and is a VL domain that is not selected for inclusion in the first polypeptide chain of the diabody), intervening spacer peptide (linker 1), capable of binding to the second table a VH domain of a monoclonal antibody (if such a second polypeptide chain contains VL _anti _- ROR1 _-VL ) or binds to ROR1 (if such a second polypeptide chain contains VL _epitope ₂ ), optionally containing half The second intervening spacer peptide (linker 2), heterodimer-promoting domain and C-terminus of the cystine residue ( Fig. 1 ).

第一多肽鏈的VL結構域與第二多肽鏈的VH結構域相互作用以形成對第一抗原(即ROR1或含有第二表位的分子)特異性的第一功能性表位-結合位點。同樣地，第二多肽鏈的VL結構域與第一多肽鏈的VH結構域相互作用，以形成對第二抗原(即，包括第二表位的分子或 ROR1)特異性的第二功能性表位-結合位點。因此，對第一和第二多肽鏈的VL和VH結構域的選擇是協調的，使得雙抗體的兩條多肽鏈總共包括能夠結合ROR1的表位和結合第二表位的VL和VH結構域(即它們總共包括VL_抗 _-ROR1-VL / VH_抗 _-ROR1-VH 和VL_表位 ₂ /VH_表位 ₂ )。The VL domain of the first polypeptide chain interacts with the VH domain of the second polypeptide chain to form a first functional epitope-binding specific for the first antigen (ie, ROR1 or a molecule containing a second epitope) Site. Likewise, the VL domain of the second polypeptide chain interacts with the VH domain of the first polypeptide chain to form a second function specific for the second antigen (ie, the molecule comprising the second epitope or ROR1) Sex epitope-binding site. Thus, the selection of the VL and VH domains of the first and second polypeptide chains is coordinated such that the two polypeptide chains of the diabody comprise a total of epitopes capable of binding ROR1 and VL and VH structures binding to the second epitope. Domains (ie, they comprise a total of VL _anti- _ROR1-VL / VH _anti- _ROR1-VH and VL _epitope ₂ / VH _epitope ₂ ).

最優選地，間插間隔體肽(即，分離這類VL和VH結構域的“連接體1”)長度被選擇，以基本上或完全地防止多肽鏈的VL和VH結構域彼此結合(例如由0、1、2、3、4、5、6、7、8或9個間插連接體氨基酸殘基組成)。因此，第一多肽鏈的VL和VH結構域基本上或完全不能彼此結合。同樣，第二多肽鏈的VL和VH結構域基本上或完全不能彼此結合。優選的間插間隔體肽(連接體1)具有序列(SEQ ID NO:33 )：GGGSGGGG。Most preferably, the intervening spacer peptide (ie, "linker 1" separating such VL and VH domains) is selected to substantially or completely prevent the VL and VH domains of the polypeptide chain from binding to each other (eg, It consists of 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9 intervening linker amino acid residues). Thus, the VL and VH domains of the first polypeptide chain are substantially or completely incapable of binding to each other. Likewise, the VL and VH domains of the second polypeptide chain are substantially or completely incapable of binding to each other. A preferred intervening spacer peptide (Linker 1) has the sequence ( SEQ ID NO: 33 ): GGGSGGGG.

基於促進這類二聚化的一個或多個多肽結構域(即，“異源二聚體促進結構域 ”)的選擇來選擇第二間插間隔體肽(連接體2)的長度和組成。典型地，第二間插間隔體肽(連接體2)包括3-20個氨基酸殘基。尤其地，當採用的異源二聚體促進結構域(一個或多個)包括/不包括半胱氨酸殘基時，使用包含半胱氨酸的第二間插間隔體肽(連接體2)。包含半胱氨酸的第二間插間隔體肽(連接體2)將包含1、2、3或更多個半胱氨酸。優選的含半胱氨酸的間隔體肽(連接體2)具有序列GGCGGG (SEQ ID NO:34 )。可選地，連接體2不包括半胱氨酸(例如GGG, GGGS (SEQ ID NO:35 )、LGGGSG (SEQ ID NO:36 )、GGGSGGGSGGG (SEQ ID NO:37 )、ASTKG (SEQ ID NO:38 )、LEPKSS (SEQ ID NO:39 )、APSSS (SEQ ID NO:40 )等)，並且使用如下所描述的含半胱氨酸的異源二聚體-促進結構域。任選地，使用含半胱氨酸的連接體2和含半胱氨酸的異源二聚體-促進結構域。The length and composition of the second intervening spacer peptide (linker 2) is selected based on the selection of one or more polypeptide domains (ie, " heterodimer promoting domains ") that facilitate such dimerization . Typically, the second intervening spacer peptide (linker 2) comprises 3-20 amino acid residues. In particular, when the heterodimer promoting domain(s) employed include/exclude a cysteine residue, a second intervening spacer peptide comprising a cysteine is used (linker 2 ). The second intervening spacer peptide (linker 2) comprising a cysteine will comprise 1, 2, 3 or more cysteines. A preferred cysteine-containing spacer peptide (linker 2) has the sequence GGCGGG ( SEQ ID NO: 34 ). Alternatively, linker 2 does not include a cysteine (eg, GGG, GGGS ( SEQ ID NO: 35 ), LGGGSG ( SEQ ID NO: 36 ), GGGSGGGSGGG ( SEQ ID NO: 37 ), ASTKG ( SEQ ID NO: 38 ), LEPKSS ( SEQ ID NO: 39 ), APSSS ( SEQ ID NO: 40 ), etc., and a cysteine-containing heterodimer-promoting domain as described below. Optionally, a cysteine-containing linker 2 and a cysteine-containing heterodimer-promoting domain are used.

異源二聚體-促進結構域在一條多肽鏈上可以是GVEPKSC (SEQ ID NO:41 )或VEPKSC (SEQ ID NO:42 )或AEPKSC (SEQ ID NO:43 )，並且在另一條多肽鏈上可以是GFNRGEC (SEQ ID NO:44 )或FNRGEC (SEQ ID NO:45 ) (US2007/0004909)。The heterodimer-promoting domain may be GVEPKSC ( SEQ ID NO: 41 ) or VEPKSC ( SEQ ID NO: 42 ) or AEPKSC ( SEQ ID NO: 43 ) on one polypeptide chain and on another polypeptide chain It may be GFNRGEC ( SEQ ID NO: 44 ) or FNRGEC ( SEQ ID NO: 45 ) (US2007/0004909).

在優選的實施方案中，異源二聚體促進結構域將包括具有相反電荷的串聯重複的螺旋結構域，例如，“E-螺旋”螺旋結構域(SEQ ID NO:46 ： E VAAL E K- E VAAL E K- E VAAL E K- E VAAL E K)，其谷氨酸殘基在pH 7形成負電荷，和“K-螺旋”結構域(SEQ ID NO:47 ： K VAAL K E- K VAAL K E- K VAAL K E- K VAAL K E)，其賴氨酸殘基在pH 7形成正電荷。這類帶電結構域的存在促進了第一和第二多肽之間的締合，因此有助於異源二聚體形成。可以使用包括上述E-螺旋和K-螺旋序列的修飾的異源二聚體促進結構域，以便包括一個或多個半胱氨酸殘基。這類半胱氨酸殘基的存在允許在一條多肽鏈上存在的螺旋與另一多肽鏈上存在的互補螺旋成為共價結合的，從而彼此共價結合多肽鏈並且增加雙抗體的穩定性。這樣的尤其優選的例子是異源二聚體促進結構域包括修飾的E-螺旋，其具有氨基酸序列 E VAA C E K- E VAAL E K- E VAAL E K- E VAAL E K (SEQ ID NO:48 )，和修飾的K-螺旋，其具有氨基酸序列 K VAA C K E- K VAAL K E- K VAAL K E- K VAAL K E (SEQ ID NO: 49 )。In a preferred embodiment, the heterodimer promoting domain will comprise a tandem repeating helical domain having an opposite charge, for example, an "E-helix" helical domain ( SEQ ID NO: 46 : E VAAL E K- E VAAL E K- E VAAL E K- E VAAL E K), whose glutamic acid residue forms a negative charge at pH 7, and the "K-helix" domain ( SEQ ID NO: 47 : K VAAL K E- K VAAL K E- K VAAL K E- K VAAL K E), whose lysine residue forms a positive charge at pH 7. The presence of such charged domains promotes association between the first and second polypeptides, thus contributing to the formation of heterodimers. A modified heterodimer promoting domain comprising the E-helix and K-helix sequences described above can be used to include one or more cysteine residues. The presence of such cysteine residues allows the helix present on one polypeptide chain to covalently bind to the complementary helix present on the other polypeptide chain, thereby covalently binding the polypeptide chain to each other and increasing the stability of the diabody. . A particularly preferred example of this is that the heterodimer promoting domain comprises a modified E-helix having the amino acid sequence E VAA C E K- E VAAL E K- E VAAL E K- E VAAL E K ( SEQ ID NO :48 ), and a modified K-helix having the amino acid sequence K VAA C K E- K VAAL K E- K VAAL K E- K VAAL K E ( SEQ ID NO: 49 ).

如WO 2012/018687中所公開的，為了提高雙抗體的體內藥物代謝動力學特性，雙抗體可被修飾，以在雙抗體的一個或多個末端處包含血清結合蛋白的多肽部分。最優選地，血清結合蛋白的這類多肽部分設置在雙抗體的多肽鏈的C-末端。白蛋白是血漿中最豐富的蛋白質並且在人中半衰期是19天。白蛋白具有若干小分子結合位點，這允許其非共價結合其他蛋白，從而延長它們的血清半衰期。鏈球菌屬(Streptococcus)菌株G148的蛋白質G的白蛋白結合結構域3 (ABD3)由形成穩定的三螺旋束的46個氨基酸殘基組成並且具有廣泛的白蛋白結合特異性(Johansson, M.U.等(2002) “Structure, Specificity, And Mode Of Interaction For Bacterial Albumin-Binding Module s,” J. Biol. Chem. 277(10):8114-8120。因此，對於改善雙抗體的體內藥物代謝動力學特性，尤其優選的血清結合蛋白的多肽部分是來自鏈球菌蛋白質G的白蛋白結合結構域(ABD)，更優選地，鏈球菌屬菌株G148的蛋白質G的白蛋白結合結構域3 (ABD3) (SEQ ID NO:50 )：LAEAKVLANR ELDKYGVSDY YKNLIDNAKS AEGVKALIDE ILAALP。As disclosed in WO 2012/018687, in order to increase the in vivo pharmacokinetic properties of the diabody, the diabody can be modified to comprise a polypeptide portion of the serum binding protein at one or more ends of the diabody. Most preferably, such a polypeptide portion of a serum binding protein is disposed at the C-terminus of the polypeptide chain of the diabody. Albumin is the most abundant protein in plasma and has a half-life of 19 days in humans. Albumin has several small molecule binding sites that allow it to bind non-covalently to other proteins, thereby extending their serum half-life. The albumin binding domain 3 (ABD3) of the protein G of Streptococcus strain G148 is composed of 46 amino acid residues forming a stable triple helix bundle and has broad albumin binding specificity (Johansson, MU et al. 2002) " Structure, Specificity, And Mode Of Interaction For Bacterial Albumin-Binding Module s," J. Biol. Chem. 277(10): 8114-8120. Therefore, in order to improve the in vivo pharmacokinetic properties of diabody, especially Preferably, the polypeptide portion of the serum binding protein is an albumin binding domain (ABD) from Streptococcal protein G, more preferably, albumin binding domain 3 (ABD3) of protein G of Streptococcus strain G148 ( SEQ ID NO) :50 ):LAEAKVLANR ELDKYGVSDY YKNLIDNAKS AEGVKALIDE ILAALP.

如WO 2012/162068 (通過引用併入本文)中公開，SEQ ID NO:50 的“去免疫化 ” 的變體具有削弱或消除II類MHC結合的能力。基於組合突變結果，對於形成這樣的去免疫化的ABD，如下取代的組合被認為是優選的取代：66D/70S +71A、66S/70S +71A、66S/70S +79A、64A/65A/71A、64A/65A/71A+66S、64A/65A/71A+66D、64A/65A/71A+66E、64A/65A/79A+66S、64A/65A/79A+66D、64A/65A/79A+66E。变异的ABD具有修饰L64A、I65A和D79A或修饰N66S、T70S和D79A。具有下述氨基酸序列： LAEAKVLANR ELDKYGVSDY YKNLI D ₆₆ NAK S ₇₀ A ₇₁ EGVKALIDE ILAALP (SEQ ID NO:51 )、或氨基酸序列： LAEAKVLANR ELDKYGVSDY YKN A ₆₄ A ₆₅ NNAKT VEGVKALI A ₇₉ E ILAALP (SEQ ID NO:52) 、或氨基酸序列： LAEAKVLANR ELDKYGVSDY YKNLI S ₆₆ NAK S ₇₀ VEGVKALI A ₇₉ E ILAALP (SEQ ID NO:53) ，的變異的去免疫化的ABD是尤其優選的，因為這類去免疫化的ABD展示基本上野生型結合，同時提供削弱的II類MHC結合。因此，具有ABD的這類雙抗體的第一多肽鏈包含第三連接體(連接體3)，其優選地位於這類多肽鏈的E螺旋(或K螺旋)結構域的C-末端，以便插在E螺旋(或K螺旋)結構域和ABD (其優選地是去免疫化的ABD)之間。用於這類連接體3的優選的序列是SEQ ID NO:35 ：GGGS。The " de-immunized " variant of SEQ ID NO: 50 has the ability to attenuate or eliminate class II MHC binding as disclosed in WO 2012/162068, which is incorporated herein by reference. Based on the combined mutation results, for the formation of such deimmunized ABD, the combination of substitutions below is considered to be a preferred substitution: 66D/70S +71A, 66S/70S +71A, 66S/70S +79A, 64A/65A/71A, 64A/65A/71A+66S, 64A/65A/71A+66D, 64A/65A/71A+66E, 64A/65A/79A+66S, 64A/65A/79A+66D, 64A/65A/79A+66E. The variant ABD has modifications to L64A, I65A and D79A or modified N66S, T70S and D79A. Has the following amino acid sequence: LAEAKVLANR ELDKYGVSDY YKNLI D ₆₆ NAK S ₇₀ A ₇₁ EGVKALIDE ILAALP ( SEQ ID NO: 51 ), or amino acid sequence: LAEAKVLANR ELDKYGVSDY YKN A ₆₄ A ₆₅ NNAKT VEGVKALI A ₇₉ E ILAALP ( SEQ ID NO: 52) , or amino acid sequence: LAEAKVLANR ELDKYGVSDY YKNLI S ₆₆ NAK S ₇₀ VEGVKALI A ₇₉ E ILAALP ( SEQ ID NO: 53) , a variant of deimmunized ABD is particularly preferred because such deimmunized ABD displays essentially Wild-type binding, while providing attenuated class II MHC binding. Thus, the first polypeptide chain of such a diabody having ABD comprises a third linker (linker 3), preferably located at the C-terminus of the E-helix (or K-helix) domain of such a polypeptide chain, such that Inserted between the E-helix (or K-helix) domain and the ABD (which is preferably a deimmunized ABD). A preferred sequence for such linker 3 is SEQ ID NO: 35 : GGGS.

C.C. 包含contain FcFc 區域的多特異性雙抗體Multispecific diabody

本發明的一個實施方案涉及能夠同時結合ROR1的表位和第二表位(即，ROR1的不同表位或不是ROR1的分子的表位)的多特異性雙抗體，其包括Fc區域。添加IgG CH2-CH3結構域至雙抗體多肽鏈之一或兩條中，以便雙抗體鏈的複合導致形成Fc區，延長了生物半衰期和/或改變了雙抗體的價。此類雙抗體包括兩條或更多條多肽鏈，其序列允許多肽鏈彼此共價結合以形成能夠同時結合ROR1的表位和第二表位的共價締合的雙抗體。將IgG CH2-CH3結構域併入到兩條雙抗體多肽上允許形成雙鏈雙特異性包含Fc-區域的雙抗體(圖 2 )。One embodiment of the invention relates to a multi-specific diabodies capable of simultaneously binding an epitope of ROR1 and a second epitope (ie, an epitope of a different epitope of ROR1 or a molecule other than ROR1), including an Fc region. The IgG CH2-CH3 domain is added to one or both of the diabody polypeptide chains such that recombination of the diabody chain results in the formation of an Fc region, prolonging the biological half-life and/or altering the valency of the diabody. Such diabody includes two or more polypeptide chains whose sequences allow the polypeptide chains to covalently bind to each other to form a covalently associated diabody capable of simultaneously binding to the epitope of ROR1 and the second epitope. Incorporation of the IgG CH2-CH3 domain onto the two diabody polypeptides allowed the formation of a double-stranded bispecific Fc-region containing diabody ( Figure 2 ).

可選地，僅僅在一條雙抗體多肽上併入IgG CH2-CH3結構域允許形成更複雜的、包含Fc區的四鏈雙特異性雙抗體(圖 3A-3C )。圖 3C 顯示具有恒定輕鏈(CL)結構域和恒定重鏈CH1結構域的代表性四鏈雙抗體，然而，可以可選地採用這類結構域的片段以及其他多肽(見，例如，圖 3A 和 3B ，美國專利公開號2013-0295121、2010-0174053和2009-0060910；歐洲專利公開號EP 2714079、EP 2601216、EP 2376109、EP 2158221；和PCT公開號WO 2012/162068、WO 2012/018687、WO 2010/080538)。因此，例如，代替CH1結構域，可採用源自人IgG的鉸鏈區的、具有氨基酸序列GVEPKSC (SEQ ID NO:41 ) VEPKSC (SEQ ID NO:42 )或AEPKSC (SEQ ID NO:43 )的肽，並且代替CL結構域，可採用人κ輕鏈的C-末端6個氨基酸GFNRGEC (SEQ ID NO:44 )或FNRGEC (SEQ ID NO:45 )。包含代表性肽的四鏈雙抗體顯示在圖 3A 中。可選地，或另外，可採用肽包括具有相反電荷的串聯螺旋結構域的肽，比如“E-螺旋”螺旋結構域 (SEQ ID NO:46 ： E VAAL E K- E VAAL E K- E VAAL E K- E VAAL E K或SEQ ID NO:48 ： E VAA C E K- E VAAL E K- E VAAL E K- E VAAL E K)；和“K-螺旋”結構域(SEQ ID NO:47 ： K VAAL K E- K VAAL K E- K VAAL K E- K VAAL K E或SEQ ID NO:49 ： K VAA C K E- K VAAL K E- K VAAL K E- K VAAL K E)。代表性的、包含螺旋結構域的四鏈雙抗體顯示在圖 3B 中。Alternatively, the incorporation of an IgG CH2-CH3 domain only on one diabody polypeptide allows for the formation of a more complex four-chain bispecific diabody comprising an Fc region ( Figures 3A-3C ). Figure 3C shows a representative four-chain diabodies having a constant light chain (CL) domain and a constant heavy chain CH1 domain, however, fragments of such domains and other polypeptides may alternatively be employed (see, for example, Figure 3A) And 3B , U.S. Patent Publication Nos. 2013-0295121, 2010-0174053, and 2009-0060910; European Patent Publication No. EP 2714079, EP 2601216, EP 2376109, EP 2158221; and PCT Publication No. WO 2012/162068, WO 2012/018687, WO 2010/080538). Thus, for example, instead of the CH1 domain, a peptide having the amino acid sequence GVEPKSC ( SEQ ID NO: 41 ) VEPKSC ( SEQ ID NO: 42 ) or AEPKSC ( SEQ ID NO: 43 ) derived from the hinge region of human IgG can be employed. And instead of the CL domain, the C-terminal 6 amino acids GFNRGEC ( SEQ ID NO: 44 ) or FNRGEC ( SEQ ID NO: 45 ) of the human kappa light chain can be used. A four-chain diabodies comprising representative peptides are shown in Figure 3A . Alternatively, or in addition, a peptide comprising a tandem helical domain having an opposite charge, such as an "E-helix" helical domain ( SEQ ID NO: 46 : E VAAL E K- E VAAL E K- E VAAL) may be employed. E K- E VAAL E K or SEQ ID NO: 48 : E VAA C E K- E VAAL E K- E VAAL E K- E VAAL E K); and the "K-helix" domain ( SEQ ID NO: 47 : K VAAL K E- K VAAL K E- K VAAL K E- K VAAL K E or SEQ ID NO: 49 : K VAA C K E- K VAAL K E- K VAAL K E- K VAAL K E). A representative four-chain diabodies comprising a helical domain are shown in Figure 3B .

本發明的含有Fc區域的分子可以包括另外的間插間隔體肽(連接體)，通常這類連接體將被併入在異源二聚體-促進結構域(例如E-螺旋或K-螺旋)和CH2-CH3結構域之間和/或在CH2-CH3結構域和可變結構域(即VH或VL)之間。通常，另外的連接體將包括3-20個氨基酸殘基，並且可任選地包括IgG鉸鏈區域(優選IgG鉸鏈區域的包含半胱氨酸的部分)的全部或一部分。可用於本發明的含Fc區域的雙特異性雙抗體分子中的連接體包括：GGGS (SEQ ID NO:35 )、LGGGSG (SEQ ID NO:36 )、GGGSGGGSGGG (SEQ ID NO:37 )、ASTKG (SEQ ID NO:38 )、LEPKSS (SEQ ID NO:39 )、APSSS (SEQ ID NO:40 )、APSSSPME (SEQ ID NO:54 )、VEPKSADKTHTCPPCP (SEQ ID NO:55 )、LEPKSADKTHTCPPCP (SEQ ID NO:56 )、DKTHTCPPCP (SEQ ID NO:57 )、GGC和GGG。為了便於克隆，可以使用LEPKSS(SEQ ID NO:39 )代替GGG或GGC。另外，氨基酸GGG或LEPKSS(SEQ ID NO:39 )之後緊接著可以是DKTHTCPPCP(SEQ ID NO:57 )，以形成可選的連接體：GGGDKTHTCPPCP(SEQ ID NO:58 )；和LEPKSSDKTHTCPPCP(SEQ ID NO:59 )。除了連接體之外或者替代連接體，本發明的含Fc區域的雙特異性分子可以併入IgG鉸鏈區域。示例性鉸鏈區包括：來自IgG1的EPKSCDKTHTCPPCP (SEQ ID NO:60 )、來自IgG2的ERKCCVECPPCP (SEQ ID NO:61 )、來自IgG3的ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP (SEQ ID NO:116 )、來自IgG4的ESKYGPPCPSCP (SEQ ID NO:62 )以及包括穩定化S228P取代(通過如Kabat中所述的EU索引編號)以減少鏈交換的IgG4鉸鏈變體ESKYGPPCP P CP (SEQ ID NO:63 )。The Fc region-containing molecule of the invention may comprise additional intervening spacer peptides (linkers), typically such linkers will be incorporated in a heterodimer-promoting domain (eg E-helix or K-helix) And between the CH2-CH3 domain and/or between the CH2-CH3 domain and the variable domain (ie VH or VL). Typically, additional linkers will include 3-20 amino acid residues, and may optionally include all or a portion of the IgG hinge region, preferably the cysteine-containing portion of the IgG hinge region. Linkers in the Fc region-containing bispecific diabody molecule useful in the present invention include: GGGS ( SEQ ID NO: 35 ), LGGGSG ( SEQ ID NO: 36 ), GGGSGGGSGGG ( SEQ ID NO: 37 ), ASTKG ( SEQ ID NO: 38 ), LEPKSS ( SEQ ID NO: 39 ), APSSS ( SEQ ID NO: 40 ), APSSSPME ( SEQ ID NO: 54 ), VEPKSADKTHTCPPCP ( SEQ ID NO: 55 ), LEPKSADKTHTCPPCP ( SEQ ID NO: 56) ), DKTHTCPPCP ( SEQ ID NO: 57 ), GGC, and GGG. For ease of cloning, LEPKSS ( SEQ ID NO: 39 ) can be used instead of GGG or GGC. Alternatively, the amino acid GGG or LEPKSS ( SEQ ID NO: 39 ) may be followed by DKTHTCPPCP ( SEQ ID NO: 57 ) to form an alternative linker: GGGDKTHTCPPCP ( SEQ ID NO: 58 ); and LEPKSSDKTHTCPPCP ( SEQ ID NO : SEQ ID NO: SEQ ID NO: 58 ) :59 ). In addition to or in lieu of a linker, an Fc region-containing bispecific molecule of the invention can be incorporated into an IgG hinge region. Exemplary hinge region comprising: EPKSCDKTHTCPPCP from IgG1, (SEQ ID NO: 60), ERKCCVECPPCP from IgG2, (SEQ ID NO: 61), derived from IgG3 in ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP (SEQ ID NO: 116) , ESKYGPPCPSCP from of IgG4 (SEQ ID NO :62 ) and the IgG4 hinge variant ESKYGPPCP P CP ( SEQ ID NO: 63 ) comprising a stabilized S228P substitution (by EU index numbering as described in Kabat) to reduce strand exchange.

如圖 3A-3C 中提供的，本發明的包含Fc區的雙特異性雙抗體可包括四條鏈。這類雙抗體的第一和第三多肽鏈包含三個結構域：(i)包含VL1的結構域、(ii)包含VH2的結構域、(iii)異源二聚體-促進結構域、和(iv)包含CH2-CH3序列的結構域。第二和第四多肽鏈包含：(i)包含VL2的結構域、(ii)包含VH1的結構域和(iii)異源二聚體-促進結構域，其中異源二聚體-促進結構域促進第一/第三多肽鏈與第二/第四多肽鏈的二聚化。第三和第四多肽鏈的VL和/或VH結構域，以及第一和第二多肽鏈的VL和/或VH結構域可以相同或不同，以便允許單特異性、雙特異性或四特異性的四價結合。符號“VL3 ”和“VH3 ”分別表示結合這類雙抗體的“第三”表位元的輕鏈可變結構域和可變重鏈結構域。類似地，符號“VL4 ”和“VH4 ”分別表示結合這類雙抗體的“第四”表位元的輕鏈可變結構域和可變重鏈結構域。本發明的包含Fc區的、代表性四鏈雙特異性雙抗體的多肽鏈的一般結構提供在表 1 中： HPD =異源二聚體-促進結構域As provided in Figures 3A-3C, the bispecific antibody comprises an Fc region of the present invention may comprise four chains. The first and third polypeptide chains of such diabody comprise three domains: (i) a domain comprising VL1, (ii) a domain comprising VH2, (iii) a heterodimer-promoting domain, And (iv) a domain comprising a CH2-CH3 sequence. The second and fourth polypeptide chains comprise: (i) a domain comprising VL2, (ii) a domain comprising VH1, and (iii) a heterodimer-promoting domain, wherein the heterodimer-promoting structure The domain facilitates dimerization of the first/third polypeptide chain with the second/fourth polypeptide chain. The VL and/or VH domains of the third and fourth polypeptide chains, and the VL and/or VH domains of the first and second polypeptide chains, may be the same or different, in order to allow monospecific, bispecific or tetra Specific tetravalent binding. The symbols " VL3 " and " VH3 " denote the light chain variable domain and the variable heavy chain domain, respectively, which bind to the "third" epitope of such a diabody. Similarly, the symbols " VL4 " and " VH4 " denote the light chain variable domain and the variable heavy chain domain, respectively, which bind to the "fourth" epitope of such a diabody. The general structure of a polypeptide chain of a representative four-chain bispecific diabody comprising an Fc region of the invention is provided in Table 1 : HPD = heterodimer-promoting domain

在具體的實施方案中，本發明的雙抗體是雙特異性的、四價(即，具有四個表位-結合位點)、包含Fc的雙抗體，其由總共四條多肽鏈構成(圖 3A-3C )。本發明的雙特異性、四價、包含Fc的雙抗體包括對於ROR1免疫特異性的兩個表位-結合位點(其可能夠結合ROR1相同的表位或ROR1不同的表位)，和對於第二分子免疫特異性的兩個表位-結合位點(其可能夠結合第二分子的相同表位或第二分子的不同表位)。優選地，第二分子是存在於效應細胞，例如T淋巴細胞、自然殺傷(NK)細胞或其他單核細胞，表面上的分子(例如CD2、CD3、CD8、CD16、T-細胞受體(TCR)、NKG2D等)。In a specific embodiment, the diabody of the invention is bispecific, tetravalent (ie, having four epitope-binding sites), an Fc-containing diabody consisting of a total of four polypeptide chains ( Fig. 3A) -3C ). The bispecific, tetravalent, Fc-containing diabody of the invention comprises two epitope-binding sites that are immunospecific for ROR1 (which may be capable of binding to the same epitope of ROR1 or a different epitope of ROR1), and The second molecule immunospecifically has two epitope-binding sites (which may be capable of binding to the same epitope of the second molecule or a different epitope of the second molecule). Preferably, the second molecule is a molecule present on the surface of an effector cell, such as a T lymphocyte, natural killer (NK) cell or other monocyte (eg, CD2, CD3, CD8, CD16, T-cell receptor (TCR) ), NKG2D, etc.).

在另一個實施方案中，本發明的包含Fc區的雙抗體可包括三條多肽鏈。這類雙抗體的第一多肽包含三個結構域：(i)包含VL1的結構域、(ii)包含VH2的結構域和(iii)包含CH2-CH3序列的結構域。這類雙抗體的第二多肽包含：(i)包含VL2的結構域、(ii)包含VH1的結構域和(iii)促進與雙抗體的第一多肽鏈異源二聚化和共價結合的結構域。這類雙抗體的第三多肽包括CH2-CH3序列。因此，這類雙抗體的第一和第二多肽鏈締合在一起，以形成能夠結合第一抗原(即， ROR1或包括第二表位的分子)的VL1/VH1表位-結合位點，以及能夠結合第二抗原(即，包括第二表位的分子或ROR1)的VL2/VH2表位-結合位點。第一和第二多肽通過涉及在它們各自的第三結構域中的半胱氨酸殘基的二硫鍵彼此結合。值得注意的是，第一和第三多肽鏈彼此複合，以形成經二硫鍵穩定的Fc區。這類雙特異性雙抗體具有增強的效價。圖 4A 和 4B 闡釋了這類雙抗體的結構。這類包含Fc區的雙抗體可具有兩個取向之一(表 2 )： HPD =異源二聚體-促進結構域In another embodiment, a diabody comprising an Fc region of the invention can comprise three polypeptide chains. The first polypeptide of such a diabody comprises three domains: (i) a domain comprising VL1, (ii) a domain comprising VH2 and (iii) a domain comprising a CH2-CH3 sequence. A second polypeptide of such a diabody comprises: (i) a domain comprising VL2, (ii) a domain comprising VH1, and (iii) promoting heterodimerization and covalent interaction of the first polypeptide chain with the diabody Combined domain. The third polypeptide of such a diabody comprises a CH2-CH3 sequence. Thus, the first and second polypeptide chains of such diabody are associated together to form a VL1/VH1 epitope-binding site capable of binding to the first antigen (ie, ROR1 or a molecule comprising a second epitope) And a VL2/VH2 epitope-binding site capable of binding to a second antigen (ie, a molecule comprising a second epitope or ROR1). The first and second polypeptides bind to each other by a disulfide bond involving a cysteine residue in their respective third domain. Notably, the first and third polypeptide chains complex with each other to form a disulfide-stabilized Fc region. Such bispecific diabodies have enhanced potency. Figures 4A and 4B illustrate the structure of such diabody. Such a diabody comprising an Fc region can have one of two orientations ( Table 2 ): HPD = heterodimer-promoting domain

在具體的實施方案中，本發明的雙抗體是雙特異性、二價(即，具有兩個表位-結合位點)、包含Fc的雙抗體，其由總共三條多肽鏈構成(圖 4A-4B )。本發明的雙特異性、二價、包含Fc的雙抗體包括對ROR1免疫特異性的一個表位-結合位點和對第二分子免疫特異性的一個表位-結合位點。優選地，第二分子是存在於效應細胞，例如T淋巴細胞、自然殺傷(NK)細胞或其他單核細胞表面上的分子(例如CD2、CD3、CD8、CD16、T-細胞受體(TCR)、NKG2D等)。In a specific embodiment, the diabody of the invention is bispecific, bivalent (ie, having two epitope-binding sites), an Fc-containing diabody consisting of a total of three polypeptide chains ( Fig. 4A- 4B ). The bispecific, bivalent, Fc-containing diabody of the invention comprises an epitope-binding site that is immunospecific for ROR1 and an epitope-binding site that is immunospecific for the second molecule. Preferably, the second molecule is a molecule present on the surface of an effector cell, such as a T lymphocyte, natural killer (NK) cell or other monocyte (eg, CD2, CD3, CD8, CD16, T-cell receptor (TCR) , NKG2D, etc.).

在進一步的實施方式中，包含Fc區的雙抗體可包括總共五條多肽鏈。在具體的實施方式中，所述五條多肽鏈的兩條具有相同的氨基酸序列。這類雙抗體的第一多肽鏈包含：(i)包含VH1的結構域、(ii)包含CH1的結構域和(iii)包含CH2-CH3序列的結構域。第一多肽鏈可以是抗體的重鏈，其包含VH1和重鏈恒定結構域。這類雙抗體的第二和第五條多肽鏈包含：(i)包含VL1的結構域和(ii)包含CL的結構域。這類雙抗體的第二和/或第五多肽鏈可以是抗體的輕鏈，其包含與第一/第三多肽鏈的VH1互補的VL1。第一、第二和/或第五多肽鏈可分離自天然產生的抗體。可選地，它們可以是重組構建的。這類雙抗體的第三多肽鏈包含：(i)包含VH1的結構域、(ii)包含CH1的結構域、(iii)包含CH2-CH3序列的結構域、(iv)包含VL2的結構域、(v)包含VH3的結構域和(vi)異源二聚體-促進結構域，其中異源二聚體-促進結構域促進第三鏈與第四鏈的二聚化。這類雙抗體的第四多肽包含：(i)包含VL3的結構域、(ii)包含VH2的結構域和(iii)促進與雙抗體的第三多肽鏈異源二聚化和共價結合的結構域。In a further embodiment, a diabody comprising an Fc region can comprise a total of five polypeptide chains. In a specific embodiment, two of the five polypeptide chains have the same amino acid sequence. The first polypeptide chain of such a diabody comprises: (i) a domain comprising VH1, (ii) a domain comprising CH1, and (iii) a domain comprising a CH2-CH3 sequence. The first polypeptide chain can be the heavy chain of an antibody comprising a VH1 and a heavy chain constant domain. The second and fifth polypeptide chains of such diabody comprise: (i) a domain comprising VL1 and (ii) a domain comprising CL. The second and/or fifth polypeptide chain of such a diabody can be the light chain of an antibody comprising a VL1 that is complementary to VH1 of the first/third polypeptide chain. The first, second and/or fifth polypeptide chains can be isolated from naturally occurring antibodies. Alternatively, they can be constructed recombinantly. The third polypeptide chain of such a diabody comprises: (i) a domain comprising VH1, (ii) a domain comprising CH1, (iii) a domain comprising a CH2-CH3 sequence, (iv) a domain comprising VL2 (v) a domain comprising VH3 and (vi) a heterodimer-promoting domain, wherein the heterodimer-promoting domain promotes dimerization of the third strand and the fourth strand. A fourth polypeptide of such a diabody comprises: (i) a domain comprising VL3, (ii) a domain comprising VH2, and (iii) a heterodimerization and covalent interaction of a third polypeptide chain that promotes diabody Combined domain.

因此，這類雙抗體的第一和第二，以及第三和第五多肽鏈締合在一起，以形成能夠結合第一表位的兩個VL1/VH1表位-結合位點。這類雙抗體的第三和第四多肽鏈締合在一起，以形成能夠結合第二表位的VL2/VH2表位-結合位點，以及能夠結合第三表位的VL3/VH3結合位點。第一和第三多肽通過涉及在它們各自恒定區中的半胱氨酸殘基的二硫鍵彼此結合。值得注意的是，第一和第三多肽鏈彼此複合，以形成Fc區。這類多特異性雙抗體具有增強的效力。圖 5 圖解了這類雙抗體的結構。應當理解，VL1/VH1、VL2/VH2和VL3/VH3結構域可以相同或不同，以便允許單特異性、雙特異性或三特異性的結合。如本文所提供的，優選地選擇這些結構域以結合ROR1的表位、第二分子的表位和任選地第三分子的表位。Thus, the first and second, and third and fifth polypeptide chains of such diabody are associated together to form two VL1/VH1 epitope-binding sites capable of binding to the first epitope. The third and fourth polypeptide chains of such diabody are associated together to form a VL2/VH2 epitope-binding site capable of binding to a second epitope, and a VL3/VH3 binding site capable of binding to a third epitope point. The first and third polypeptides bind to each other by a disulfide bond involving a cysteine residue in their respective constant regions. Notably, the first and third polypeptide chains complex with each other to form an Fc region. Such multispecific diabodies have enhanced potency. Figure 5 illustrates the structure of such diabody. It will be appreciated that the VL1/VH1, VL2/VH2 and VL3/VH3 domains may be the same or different in order to allow for monospecific, bispecific or trispecific binding. As provided herein, these domains are preferably selected to bind to an epitope of ROR1, an epitope of a second molecule, and optionally an epitope of a third molecule.

選擇多肽鏈的VL和VH結構域，以便形成對於期望的表位特異性的VL/VH結合位點。通過多肽鏈的締合形成的VL/VH結合位點可以相同或不同，以便允許單特異性、雙特異性、三特異性或四特異性的四價結合。尤其地，可選擇VL和VH結構域，以便多價雙抗體可包括針對第一表位的兩個結合位點和針對第二表位的兩個結合位點，或針對第一表位的三個結合位點和針對第二表位的一個結合位點，或針對第一表位的兩個結合位點，針對第二表位的一個結合位點和針對第三表位的一個結合位點(如圖 5 中所描繪)。本發明的代表性包含Fc區的五鏈雙抗體的多肽鏈的一般結構提供在表 3 中： HPD =異源二聚體-促進結構域The VL and VH domains of the polypeptide chain are selected to form a VL/VH binding site that is specific for the desired epitope. The VL/VH binding sites formed by the association of polypeptide chains may be the same or different to allow for monospecific, bispecific, trispecific or tetraspecific tetravalent binding. In particular, the VL and VH domains can be selected such that the multivalent diabody can comprise two binding sites for the first epitope and two binding sites for the second epitope, or three for the first epitope Binding sites and one binding site for the second epitope, or two binding sites for the first epitope, one binding site for the second epitope and one binding site for the third epitope (as depicted in FIG. 5). A typical structure of a representative polypeptide chain of a five-chain diabodies comprising an Fc region of the invention is provided in Table 3 : HPD = heterodimer-promoting domain

在具體的實施方式中，本發明的雙抗體是雙特異性、四價(即，具有四個表位-結合位點)、包含Fc的雙抗體，其由總共五條多肽鏈構成，具有對於ROR1免疫特異性的兩個表位-結合位點(其可能夠結合ROR1的相同表位或ROR1的不同表位)，和對於第二分子特異性的兩個表位-結合位點(其可能夠結合第二分子的相同表位或第二分子的不同表位)。在另一實施方式中，本發明的雙特異性、四價、包含Fc的雙抗體包括對於ROR1免疫特異性的三個表位-結合位點(其可能夠結合ROR1的相同的表位或ROR1的兩個或三個不同的表位)，和對於第二分子特異性的一個表位-結合位點。在另一實施方式中，本發明的雙特異性、四價、包含Fc的雙抗體包括對於ROR1免疫特異性的一個表位-結合位點，和對於第二分子特異性的三個表位-結合位點(其可能夠結合第二分子的相同的表位或第二分子的兩個或三個不同的表位)。如上所述，可以選擇VL和VH結構域以允許三特異性結合。因此，本發明還包括三特異性、四價、含Fc的雙抗體。本發明的三特異性、四價、含Fc的雙抗體包括對ROR1免疫特異性的兩個表位-結合位點、對第二分子免疫特異性的一個表位-結合位點和對第三分子免疫特異性的一個表位-結合位點。在某些實施方案中，第二分子是存在於效應細胞例如T淋巴細胞、自然殺傷(NK)細胞或其他單核細胞表面上的分子(例如CD2、CD3、CD8、CD16、T細胞受體(TCR)、NKG2D等)。在某些實施方案中，第二分子是CD3，第三分子是CD8。In a specific embodiment, the diabody of the invention is bispecific, tetravalent (ie, having four epitope-binding sites), an Fc-containing diabody consisting of a total of five polypeptide chains with ROR1 Immunospecific two epitope-binding sites (which may be capable of binding to the same epitope of ROR1 or different epitopes of ROR1), and two epitope-binding sites specific for the second molecule (which may be capable of Binding to the same epitope of the second molecule or to a different epitope of the second molecule). In another embodiment, the bispecific, tetravalent, Fc-containing diabody of the invention comprises three epitope-binding sites that are immunospecific for ROR1 (which may be capable of binding to the same epitope of ROR1 or ROR1 Two or three different epitopes), and one epitope-binding site specific for the second molecule. In another embodiment, the bispecific, tetravalent, Fc-containing diabody of the invention comprises an epitope-binding site that is immunospecific for ROR1, and three epitopes specific for the second molecule - A binding site (which may be capable of binding to the same epitope of the second molecule or two or three different epitopes of the second molecule). As described above, the VL and VH domains can be selected to allow for trispecific binding. Thus, the invention also encompasses trispecific, tetravalent, Fc-containing diabody. The trispecific, tetravalent, Fc-containing diabody of the invention comprises two epitope-binding sites that are immunospecific for ROR1, an epitope-binding site that is immunospecific for the second molecule, and a third An epitope-binding site specific for molecular immunity. In certain embodiments, the second molecule is a molecule that is present on the surface of an effector cell, such as a T lymphocyte, natural killer (NK) cell, or other monocyte (eg, CD2, CD3, CD8, CD16, T cell receptor ( TCR), NKG2D, etc.). In certain embodiments, the second molecule is CD3 and the third molecule is CD8.

D.D. 含有contain FcFc 區域的三價結合分子Trivalent binding molecule

本發明的另一個實施方案涉及包括Fc區域的三價結合分子，其能夠同時結合第一表位、第二表位和第三表位，其中至少一個這樣的表位與另一個不同。這類三價結合分子包括三個表位-結合位點，其中兩個是雙抗體型結合結構域，其提供結合位點A和結合位點B，並且其中一個是Fab型結合結構域或scFv型結合結構域，其提供結合位點C (見，例如，圖 6A-6F ，和PCT公佈號：PCT/US15/33081；和PCT/US15/33076)。這類三價結合分子因此包括能夠結合第一表位的“VL1 ”/“VH1 ”結構域和能夠結合第二表位的“VL2 ”/“VH2 ”結構域和能夠結合這類三價結合分子的“第三”表位的“VL3 ”和“VH3 ”結構域。“雙抗體型結合結構域”是雙抗體，尤其是DART®雙抗體中存在的表位-結合位點的類型，如上所述。每個“Fab型結合結構域”和“scFv型結合結構域”是通過免疫球蛋白輕鏈的VL結構域和免疫球蛋白重鏈的互補VH結構域相互作用形成的表位-結合位點。Fab型結合結構域與雙抗體型結合結構域的差異在於形成Fab型結合結構域的兩條多肽鏈僅僅包括單個表位-結合位點，而形成雙抗體型結合結構域的兩條多肽鏈包括至少兩個表位-結合位點。類似地，scFv型結合結構域與雙抗體型結合結構域的差異也在於它們僅僅包括單個表位-結合位點。因此，如本文使用的Fab型和scFv型結合結構域與雙抗體型結合結構域不同。Another embodiment of the invention relates to a trivalent binding molecule comprising an Fc region capable of simultaneously binding a first epitope, a second epitope and a third epitope, wherein at least one such epitope is different from the other. Such trivalent binding molecules include three epitope-binding sites, two of which are diabody-type binding domains that provide binding site A and binding site B, and one of which is a Fab-type binding domain or scFv A binding domain that provides a binding site C (see, for example, Figures 6A-6F , and PCT Publication No.: PCT/US15/33081; and PCT/US15/33076). Such trivalent binding molecules thus include a " VL1 "/" VH1 " domain capable of binding to a first epitope and a " VL2 "/" VH2 " domain capable of binding to a second epitope and are capable of binding such trivalent binding molecules The " VL3 " and " VH3 " domains of the "third" epitope. A "diabody-type binding domain" is a type of epitope-binding site present in a diabody, particularly a DART® diabody, as described above. Each "Fab-type binding domain" and "scFv-type binding domain" is an epitope-binding site formed by the interaction of the VL domain of an immunoglobulin light chain and the complementary VH domain of an immunoglobulin heavy chain. The Fab-type binding domain differs from the diabody-type binding domain in that the two polypeptide chains forming the Fab-type binding domain comprise only a single epitope-binding site, while the two polypeptide chains forming the diabody-type binding domain include At least two epitope-binding sites. Similarly, the scFv-type binding domain differs from the diabody-type binding domain in that they only comprise a single epitope-binding site. Thus, the Fab-type and scFv-type binding domains as used herein differ from the diabody-type binding domain.

通常，本發明的三價結合分子包括四條不同的多肽鏈(見圖 6A-6B )，但是，例如，通過彼此融合這類多肽鏈(例如，經肽鍵)或通過分開這類多肽鏈，以形成另外的多肽鏈，或通過經二硫鍵締合更少的或另外的多肽鏈，分子可包括更少或更多數量的多肽鏈。圖 6C-6F 通過示意性描繪具有三條多肽鏈的這類分子闡釋了本發明的這個方面。如圖 6A-6F 中提供的，本發明的三價結合分子可具有可選的取向，其中雙抗體型結合結構域在Fc區的N-末端(圖 6A 、6C 和6D )或C-末端(圖 6B 、6E 和6F )。Typically, a trivalent binding molecule of the invention comprises four different polypeptide chains (see FIGS. 6A-6B), but, for example, by such a fusion polypeptide chain with one another (e.g., via a peptide bond) or by a separate polypeptide chains such as to Additional polypeptide chains are formed, or by associating fewer or additional polypeptide chains through a disulfide bond, the molecule can include fewer or greater numbers of polypeptide chains. Figures 6C-6F illustrate this aspect of the invention by schematically depicting such molecules having three polypeptide chains. Figure 6A-6F provided, trivalent binding molecules of the present invention may have optional orientation, wherein diabody type binding domains at the N- terminus of the Fc region (FIG. 6A, 6C and 6D) or the C- terminus ( Figures 6B , 6E and 6F ).

在某些實施方案中，本發明的這類三價結合分子的第一多肽鏈包含：(i)包含VL1的結構域、(ii)包含VH2的結構域、(iii)異源二聚體-促進結構域和(iv)包含CH2-CH3序列的結構域。VL1和VL2結構域位於包含CH2-CH3的結構域的N-末端或C-末端，如表 4 (也見圖 6A 和6B )中所顯示。這類實施方式的第二多肽鏈包含：(i)包含VL2的結構域、(ii)包含VH1的結構域和(iii)異源二聚體-促進結構域。這類實施方式的第三多肽鏈包含：(i)包含VH3的結構域、(ii)包含CH1的結構域和(iii)包含CH2-CH3序列的結構域。第三多肽鏈可以是包含VH3和重鏈恒定區的抗體的重鏈，或包含這樣的結構域的多肽。這類實施方式的第四多肽包含：(i)包含VL3的結構域和(ii)包含CL的結構域。第四多肽鏈可以是包含與第三多肽鏈的VH3互補的VL3的抗體的輕鏈，或包含這樣的結構域的多肽。第三或第四多肽鏈可分離自天然產生的抗體。可選地，它們可經重組、合成通過其方式而構建。In certain embodiments, a first polypeptide chain of such a trivalent binding molecule of the invention comprises: (i) a domain comprising VL1, (ii) a domain comprising VH2, (iii) a heterodimer a promoting domain and (iv) a domain comprising a CH2-CH3 sequence. N- or C- terminus located VL1 and VL2 domain comprising the CH2-CH3 domain as shown in Table 4 (see also FIGS. 6A and 6B) are shown. The second polypeptide chain of such embodiments comprises: (i) a domain comprising VL2, (ii) a domain comprising VH1, and (iii) a heterodimer-promoting domain. A third polypeptide chain of this type of embodiment comprises: (i) a domain comprising VH3, (ii) a domain comprising CH1, and (iii) a domain comprising a CH2-CH3 sequence. The third polypeptide chain can be a heavy chain of an antibody comprising VH3 and a heavy chain constant region, or a polypeptide comprising such a domain. A fourth polypeptide of such an embodiment comprises: (i) a domain comprising VL3 and (ii) a domain comprising CL. The fourth polypeptide chain may be a light chain of an antibody comprising VL3 complementary to VH3 of the third polypeptide chain, or a polypeptide comprising such a domain. The third or fourth polypeptide chain can be isolated from naturally occurring antibodies. Alternatively, they can be constructed by recombination and synthesis.

第一和第二多肽鏈的輕鏈可變結構域通過間插間隔體肽與這類多肽鏈的重鏈可變結構域分開，所述間插間隔體肽長度太短而不允許它們的VL1/VH2 (或它們的VL2/VH1)結構域締合在一起形成能夠結合第一或第二表位的表位結合位點。用於該目的的優選間插間隔體肽(連接體1)具有序列(SEQ ID NO: 33 )：GGGSGGGG。三價結合分子的其他結構域可通過任選地包括半胱氨酸殘基的一個或多個間插間隔體肽(連接體)分開。尤其地，如上面提供，這類連接體通常被併入在可變結構域(即，VH或VL)和肽異源二聚體促進結構域(例如，E-螺旋或K-螺旋)之間和這類肽異源二聚體促進結構域(例如，E-螺旋或K-螺旋)和CH2-CH3結構域之間。上面提供了用於產生三價結合分子的示例性連接體，並且其也提供在PCT公佈號：PCT/US15/33081和PCT/US15/33076中。因此，這類三價結合分子的第一和第二多肽鏈締合在一起，形成能夠結合第一表位的VL1/VH1結合位點，以及能夠結合第二表位的VL2/VH2結合位點。這類三價結合分子的第三和第四多肽鏈締合在一起而形成能夠結合第三表位的VL3/VH3結合位點。The light chain variable domains of the first and second polypeptide chains are separated from the heavy chain variable domains of such polypeptide chains by intervening spacer peptides that are too short in length to allow their The VL1/VH2 (or their VL2/VH1) domains are joined together to form an epitope binding site capable of binding to the first or second epitope. A preferred intervening spacer peptide (Linker 1) for this purpose has the sequence ( SEQ ID NO: 33 ): GGGSGGGG. Other domains of the trivalent binding molecule can be separated by one or more intervening spacer peptides (linkers), optionally including a cysteine residue. In particular, as provided above, such linkers are typically incorporated between a variable domain (ie, VH or VL) and a peptide heterodimer promoting domain (eg, an E-helix or a K-helix) And such peptide heterodimers promote a domain (eg, E-helix or K-helix) and a CH2-CH3 domain. Exemplary linkers for the production of trivalent binding molecules are provided above, and are also provided in PCT Publication Nos.: PCT/US15/33081 and PCT/US15/33076. Thus, the first and second polypeptide chains of such a trivalent binding molecule are associated together to form a VL1/VH1 binding site capable of binding to the first epitope, and a VL2/VH2 binding site capable of binding to the second epitope. point. The third and fourth polypeptide chains of such trivalent binding molecules are associated together to form a VL3/VH3 binding site capable of binding to a third epitope.

如上所述，本發明的三價結合分子可包括三條多肽。包括三條多肽鏈的三價結合分子可通過將第四多肽N-末端的結構域連接至第三多肽的包含VH3的結構域(例如，使用間插間隔體肽(連接體 4 ))而獲得。可選地，使用包含下述結構域的本發明的三價結合分子的第三多肽鏈：(i)包含VL3的結構域、(ii)包含VH3的結構域和(iii)包含CH2-CH3序列的結構域，其中VL3和VH3通過間插間隔體肽彼此間隔開，所述間插間隔體肽足夠長(至少9個或更多個氨基酸殘基)，以便允許這些結構域的締合形成表位-結合位點。對於該目的，一個優選的間插間隔體肽具有序列：GGGGSGGGGSGGGGS (SEQ ID NO:64 )。As described above, the trivalent binding molecule of the present invention may comprise three polypeptides. A trivalent binding molecule comprising three polypeptide chains can be joined by linking the N-terminal domain of the fourth polypeptide to a VH3-containing domain of the third polypeptide (eg, using an intervening spacer peptide ( linker 4 )) obtain. Alternatively, a third polypeptide chain of a trivalent binding molecule of the invention comprising: (i) a domain comprising VL3, (ii) a domain comprising VH3 and (iii) comprising CH2-CH3 is used a domain of a sequence in which VL3 and VH3 are separated from each other by an intervening spacer peptide that is sufficiently long (at least 9 or more amino acid residues) to allow association of these domains Epitope-binding site. For this purpose, a preferred intervening spacer peptide has the sequence: GGGGSGGGGSGGGGS ( SEQ ID NO: 64 ).

應當理解，這類三價結合分子的VL1/VH1、VL2/VH2和VL3/VH3結構域可以不同，以便允許單特異性、雙特異性或三特異性的結合。具體地，可以選擇VL和VH結構域，使得三價結合分子包括針對第一表位的兩個結合位點和針對第二表位的一個結合位點，或針對第一表位的一個結合位點和針對第二表位的兩個結合位點，或者針對第一表位的一個結合位點，針對第二表位的一個結合位點和針對第三表位的一個結合位點。It will be appreciated that the VL1/VH1, VL2/VH2 and VL3/VH3 domains of such trivalent binding molecules may differ to allow for monospecific, bispecific or trispecific binding. Specifically, the VL and VH domains can be selected such that the trivalent binding molecule comprises two binding sites for the first epitope and one binding site for the second epitope, or one binding site for the first epitope Point and two binding sites for the second epitope, or one binding site for the first epitope, one binding site for the second epitope and one binding site for the third epitope.

然而，如本文所提供的，優選地選擇這些結構域，以便結合ROR1的表位、第二分子的表位和第三分子的表位。在某些實施方案中，第二分子是存在於效應細胞例如T淋巴細胞、自然殺傷(NK)細胞或其他單核細胞表面上的分子(例如CD2、CD3、CD8、CD16、T細胞受體(TCR)、NKG2D等)。在某些實施方案中，第三分子是CD8。However, as provided herein, these domains are preferably selected to bind to the epitope of ROR1, the epitope of the second molecule, and the epitope of the third molecule. In certain embodiments, the second molecule is a molecule that is present on the surface of an effector cell, such as a T lymphocyte, natural killer (NK) cell, or other monocyte (eg, CD2, CD3, CD8, CD16, T cell receptor ( TCR), NKG2D, etc.). In certain embodiments, the third molecule is CD8.

本發明的代表性三價結合分子的多肽鏈的一般結構被提供在圖 6A-6F 和表 4 中： HPD =異源二聚體-促進結構域The general structure of the polypeptide chain of a representative trivalent binding molecule of the invention is provided in Figures 6A-6F and Table 4 : HPD = heterodimer-promoting domain

本發明的一個實施方案涉及三價結合分子，其包括針對ROR1的兩個表位-結合位點和針對第二分子的一個表位-結合位點。針對ROR1的兩個表位-結合位點可結合相同的表位或不同的表位。本發明的另一實施方式涉及三價結合分子，其包括針對ROR1的一個表位-結合位點和針對第二分子的兩個表位-結合位點。針對第二分子的兩個表位-結合位點可結合第二分子的相同的表位或不同的表位。本發明的另一個實施方案涉及三特異性三價結合分子，其包括針對ROR1的一個表位-結合位點，針對第二分子的一個表位結合位點和針對第三分子的一個表位結合位點的。在某些實施方案中，第二分子是存在於效應細胞例如T淋巴細胞、自然殺傷(NK)細胞或其他單核細胞表面上的分子(例如CD2、CD3、CD8、CD16、T細胞受體(TCR)、NKG2D等)。在某些實施方案中，第二分子是CD3，第三分子是CD8。如上所述，這樣的三價結合分子可以包括三條、四條、五個或更多條多肽鏈。One embodiment of the invention relates to a trivalent binding molecule comprising two epitope-binding sites for ROR1 and one epitope-binding site for a second molecule. The two epitope-binding sites for ROR1 can bind to the same epitope or different epitopes. Another embodiment of the invention relates to a trivalent binding molecule comprising an epitope-binding site for ROR1 and two epitope-binding sites for a second molecule. The two epitope-binding sites for the second molecule can bind to the same epitope or different epitopes of the second molecule. Another embodiment of the invention relates to a trispecific binding molecule comprising an epitope-binding site for ROR1, an epitope binding site for a second molecule and an epitope binding for a third molecule The location of the site. In certain embodiments, the second molecule is a molecule that is present on the surface of an effector cell, such as a T lymphocyte, natural killer (NK) cell, or other monocyte (eg, CD2, CD3, CD8, CD16, T cell receptor ( TCR), NKG2D, etc.). In certain embodiments, the second molecule is CD3 and the third molecule is CD8. As noted above, such trivalent binding molecules can include three, four, five or more polypeptide chains.

VII.VII. 恒定結構域和變異的Constant domain and variant FcFc 區域region

本文提供了可用於生成本發明的ROR1-結合分子(例如抗體、雙抗體、三價結合分子等)的抗體“恒定結構域”。Provided herein are antibody "constant domains" that can be used to generate ROR1-binding molecules of the invention (eg, antibodies, diabodies, trivalent binding molecules, etc.).

優選的CL結構域是人IgG CL κ結構域。示例性人CL κ結構域的氨基酸序列是(SEQ ID NO:65 )： RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGECA preferred CL domain is a human IgG CL κ domain. The amino acid sequence of an exemplary human CL κ domain is ( SEQ ID NO: 65 ): RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

可選地，示例性CL結構域是人IgG CL λ結構域。示例性人CL λ結構域的氨基酸序列是(SEQ ID NO:66 )： QPKAAPSVTL FPPSSEELQA NKATLVCLIS DFYPGAVTVA WKADSSPVKA GVETTPSKQS NNKYAASSYL SLTPEQWKSH RSYSCQVTHE GSTVEKTVAP TECSAlternatively, an exemplary CL domain is a human IgG CL λ domain. The amino acid sequence of an exemplary human CL λ domain is ( SEQ ID NO: 66 ): QPKAAPSVTL FPPSSEELQA NKATLVCLIS DFYPGAVTVA WKADSSPVKA GVETTPSKQS NNKYAASSYL SLTPEQWKSH RSYSCQVTHE GSTVEKTVAP TECS

如本文所提供的，本發明的ROR1-結合分子可以包括Fc區域。本發明的這些分子的Fc區域可以是任何同種型(例如，IgG1、IgG2、IgG3或IgG4)。本發明的ROR1-結合分子還可以包括CH1結構域和/或鉸鏈區域。當存在時，CH1結構域和/或鉸鏈區域可以是任何同種型(例如，IgG1、IgG2、IgG3或IgG4)，並且優選與期望的Fc區域屬於相同的同種型。As provided herein, a ROR1-binding molecule of the invention can include an Fc region. The Fc region of these molecules of the invention may be of any isotype (eg, IgGl, IgG2, IgG3 or IgG4). The ROR1-binding molecules of the invention may also include a CH1 domain and/or a hinge region. When present, the CH1 domain and/or hinge region can be of any isotype (eg, IgG1, IgG2, IgG3, or IgG4), and preferably belongs to the same isotype as the desired Fc region.

示例性CH1結構域是人IgG1 CH1結構域。示例性人IgG1 CH1結構域的氨基酸序列是(SEQ ID NO:67 )： ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVAn exemplary CH1 domain is the human IgG1 CH1 domain. The amino acid sequence of the exemplary human IgG1 CH1 domain is ( SEQ ID NO: 67 ): ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRV

示例性CH1結構域是人IgG2 CH1結構域。示例性人IgG2 CH1結構域的氨基酸序列是(SEQ ID NO:68 )： ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTVAn exemplary CH1 domain is the human IgG2 CH1 domain. The amino acid sequence of the exemplary human IgG2 CH1 domain is ( SEQ ID NO: 68 ): ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTV

示例性CH1結構域是人IgG3 CH1結構域。示例性人IgG3 CH1結構域的氨基酸序列是(SEQ ID NO:117 )： ASTKGPSVFP LAPCSRSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YTCNVNHKPS NTKVDKRVAn exemplary CH1 domain is the human IgG3 CH1 domain. The amino acid sequence of the exemplary human IgG3 CH1 domain is ( SEQ ID NO: 117 ): ASTKGPSVFP LAPCSRSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YTCNVNHKPS NTKVDKRV

示例性CH1結構域是人IgG4 CH1結構域。示例性人IgG4 CH1結構域的氨基酸序列是(SEQ ID NO:69 )： ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVAn exemplary CH1 domain is the human IgG4 CH1 domain. The amino acid sequence of the exemplary human IgG4 CH1 domain is ( SEQ ID NO: 69 ): ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRV

示例性鉸鏈區域是人IgG1鉸鏈區域。示例性人IgG1鉸鏈區域的氨基酸序列是(SEQ ID NO:60 )：EPKSCDKTHTCPPCP。An exemplary hinge region is the human IgGl hinge region. The amino acid sequence of the exemplary human IgGl hinge region is ( SEQ ID NO: 60 ): EPKSCDKTHTCPPCP.

另一個示例性鉸鏈區域是人IgG2鉸鏈區域。示例性人IgG2鉸鏈區域的氨基酸序列是(SEQ ID NO:61 )：ERKCCVECPPCP。Another exemplary hinge region is the human IgG2 hinge region. The amino acid sequence of the exemplary human IgG2 hinge region is ( SEQ ID NO: 61 ): ERKCCVECPPCP.

另一個示例性鉸鏈區域是人IgG3鉸鏈區域。示例性人IgG3鉸鏈區域的氨基酸序列是(SEQ ID NO:116 )： ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP。Another exemplary hinge region is the human IgG3 hinge region. The amino acid sequence of the exemplary human IgG3 hinge region is ( SEQ ID NO: 116 ): ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP.

另一個示例性鉸鏈區域是人IgG4鉸鏈區域。示例性人IgG4鉸鏈區域的氨基酸序列是(SEQ ID NO:62 )：ESKYGPPCPSCP。如本文所述，IgG4鉸鏈區域可以包括穩定化突變，例如S228P取代。示例性穩定的IgG4鉸鏈區域的氨基酸序列是(SEQ ID NO:63 )：ESKYGPPCPPCP。Another exemplary hinge region is the human IgG4 hinge region. The amino acid sequence of an exemplary human IgG4 hinge region is ( SEQ ID NO: 62 ): ESKYGPP CPSCP. As described herein, the IgG4 hinge region can include a stabilizing mutation, such as a S228P substitution. The amino acid sequence of an exemplary stable IgG4 hinge region is ( SEQ ID NO: 63 ): ESKYGPPCPPCP.

本發明的包含Fc區的分子(例如，抗體、雙抗體、三價結合分子等)的Fc區可以是完整的Fc區(例如，完整的IgG Fc區)或僅僅Fc區的片段。任選地，本發明的包含Fc區的分子的Fc區缺少C-末端賴氨酸氨基酸殘基。The Fc region of a Fc region-containing molecule (e.g., antibody, diabody, trivalent binding molecule, etc.) of the invention can be a complete Fc region (e.g., an intact IgG Fc region) or a fragment of only the Fc region. Optionally, the Fc region of a molecule comprising an Fc region of the invention lacks a C-terminal lysine amino acid residue.

在傳統免疫功能中，抗體-抗原複合物與免疫系統的細胞的相互作用產生各種各樣的應答，範圍從效應子功能，比如抗體依賴性細胞毒性、肥大細胞脫粒和吞噬，到免疫調節信號，比如調節淋巴細胞增殖和抗體分泌。所有這些相互作用通過抗體或免疫複合物的Fc區與造血細胞上專用細胞表面受體的結合來啟動。由抗體和免疫複合物引發的細胞應答的多樣性由三種Fc受體：FcγRI (CD64)、FcγRII (CD32)和FcγRIII (CD16)的結構異質性造成。FcγRI (CD64)、FcγRIIA (CD32A)和FcγRIII (CD16)是啟動(即，免疫系統增強)性受體；FcγRIIB (CD32B)是抑制(即，免疫系統抑制)性受體。另外，與新生Fc受體(FcRn)的相互作用介導IgG分子從內體至細胞表面的再迴圈並且釋放進入血液中。上面呈現了示例性野生型IgG1 (SEQ ID NO:1 )、IgG2 (SEQ ID NO:2 )、IgG3 (SEQ ID NO:3 )和IgG4 (SEQ ID NO:4 )的氨基酸序列。In traditional immune functions, the interaction of antibody-antigen complexes with cells of the immune system produces a variety of responses ranging from effector functions such as antibody-dependent cytotoxicity, mast cell degranulation and phagocytosis, to immunomodulatory signals, For example, regulation of lymphocyte proliferation and antibody secretion. All of these interactions are initiated by the binding of the Fc region of an antibody or immune complex to a specialized cell surface receptor on hematopoietic cells. The diversity of cellular responses elicited by antibodies and immune complexes is caused by the structural heterogeneity of the three Fc receptors: FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). FcγRI (CD64), FcγRIIA (CD32A) and FcγRIII (CD16) are priming (ie, immune system-enhancing) receptors; FcγRIIB (CD32B) is an inhibitory (ie, immune system inhibitor) receptor. In addition, interaction with the neonatal Fc receptor (FcRn) mediates recirculation of the IgG molecule from the endosome to the cell surface and release into the blood. The amino acid sequences of exemplary wild-type IgG1 ( SEQ ID NO: 1 ), IgG2 ( SEQ ID NO: 2 ), IgG3 ( SEQ ID NO: 3 ), and IgG4 ( SEQ ID NO: 4 ) are presented above.

Fc區域的修飾可以導致改變的表型，例如改變的血清半衰期、改變的穩定性、改變的對細胞酶的易感性或改變的效應子功能。因此，就效應子功能而言，可期望修飾本發明的包含Fc區的ROR1-結合分子，例如，以便增強這類分子治療癌症的效力。在某些情況下，例如在作用機制涉及阻斷或拮抗，但是不殺傷攜帶靶抗原的細胞的抗體的情況下，期望降低或消除效應子功能。當涉及非期望的細胞，比如腫瘤和外源細胞時，其中FcγRs以低水準表達，例如，具有低水準的FcγRIIB腫瘤特異性B細胞(例如，非霍奇金淋巴瘤、CLL和伯基特淋巴瘤)，一般期望增加的效應子功能。本發明的分子具有這樣的賦予的或改變的效應子功能活性，可用於治療和/或預防其中期望增強的效應子功能活性效力的疾病、病症或感染。Modification of the Fc region can result in altered phenotypes, such as altered serum half-life, altered stability, altered susceptibility to cellular enzymes, or altered effector function. Thus, in terms of effector function, it may be desirable to modify an ROR1-binding molecule comprising an Fc region of the invention, for example, to enhance the efficacy of such molecules in treating cancer. In certain instances, such as where the mechanism of action involves blocking or antagonizing, but does not kill antibodies to cells carrying the target antigen, it is desirable to reduce or eliminate effector function. When it comes to undesired cells, such as tumors and foreign cells, FcγRs are expressed at low levels, for example, with low levels of FcγRIIB tumor-specific B cells (eg, non-Hodgkin's lymphoma, CLL, and Burkitt's lymph) Tumors), generally expected to increase the effector function. The molecules of the invention have such conferred or altered effector functional activity and are useful in the treatment and/or prevention of diseases, disorders or infections in which it is desired to enhance the potency of effector functional activity.

因此，在某些實施方案中，本發明的包含Fc區的分子的Fc區可以是工程化的變異的Fc區。儘管本發明雙特異性包含Fc區的分子的Fc區可以具有結合一個或多個Fc受體(例如，FcγR)的能力，但是更優選地這類變異的Fc區具有對FcγRIA (CD64)、FcγRIIA (CD32A)、FcγRIIB (CD32B)、FcγRIIIA (CD16a)或FcγRIIIB (CD16b)改變的結合(相對於野生型Fc區展示的結合)，例如，具有與啟動性受體增強的結合和/或具有對抑制性受體基本上降低的結合能力或沒有結合抑制性受體的能力。因此，本發明包含Fc區的分子的Fc區可包括完整的Fc區的一些或所有CH2結構域和/或一些或所有CH3結構域，或可包括變異的CH2和/或變異的CH3序列(其相對於完整的Fc區的CH2或CH3結構域，可包括，例如，一個或多個***和/或一個或多個缺失)。這類Fc區可包括非Fc多肽部分，或可包括非天然完整Fc區的部分，或可包括非天然產生的取向的CH2和/或CH3結構域(比如，例如，兩個CH2結構域或兩個CH3結構域，或者在N-末端至C-末端方向上，連接至CH2結構域的CH3結構域等)。Thus, in certain embodiments, the Fc region of a molecule comprising an Fc region of the invention can be an engineered variant Fc region. Although the Fc region of a bispecific molecule comprising an Fc region of the invention may have the ability to bind one or more Fc receptors (eg, FcγR), it is more preferred that such mutated Fc regions have a FcγRIA (CD64), FcγRIIA Altered binding (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a) or FcγRIIIB (CD16b) (binding relative to the display of the wild-type Fc region), for example, has enhanced binding to the priming receptor and/or has inhibition Sexual receptors have substantially reduced binding capacity or the ability to bind to inhibitory receptors. Thus, an Fc region of a molecule comprising an Fc region of the invention may comprise some or all of the CH2 domain of the entire Fc region and/or some or all of the CH3 domain, or may comprise a mutated CH2 and/or variant CH3 sequence (which The CH2 or CH3 domain relative to the entire Fc region may include, for example, one or more insertions and/or one or more deletions). Such Fc regions can include non-Fc polypeptide portions, or can include portions of a non-native intact Fc region, or can include non-naturally occurring oriented CH2 and/or CH3 domains (eg, for example, two CH2 domains or two CH3 domains, or in the N-terminal to C-terminal direction, to the CH3 domain of the CH2 domain, etc.).

被鑒定為改變效應子功能的Fc區修飾是本領域已知的，包括增加與啟動性受體(例如，FcγRIIA (CD16A)的結合的修飾和減少與抑制性受體(例如，FcγRIIB (CD32B)結合的修飾(例如，FcγRIIB (CD32B) (見，例如，Stavenhagen, J.B.等(2007) “Fc Optimization Of Therapeutic Antibodies Enhances Their Ability To Kill Tumor Cells In Vitro And Controls Tumor Expansion In Vivo Via Low-Affinity Activating Fcgamma Receptors ,” Cancer Res. 57(18):8882-8890)。表 5 列舉了示例性修飾的示例性單、雙、三、四和五取代(編號和取代相對於SEQ ID NO:1 的氨基酸序列)，其增加了與啟動性受體的結合和/或降低了與抑制性受體的結合。 Fc region modifications identified as altering effector functions are known in the art, including modifications that increase binding to activating receptors (e.g., FcyRIIA (CD16A) and reduced and inhibitory receptors (e.g., FcyRIIB (CD32B)) Binding modifications (eg, FcyRIIB (CD32B) (see, eg, Stavenhagen, JB et al. (2007) " Fc Optimization Of Therapeutic Antibodies Enhances Their Ability To Kill Tumor Cells In Vitro And Controls Tumor Expansion In Vivo Via Low-Affinity Activating Fcgamma Receptors , Cancer Res. 57(18): 8882-8890. Table 5 lists exemplary single, double, triple, tetra and penta substitutions of the exemplary modifications (numbering and substitution relative to the amino acid sequence of SEQ ID NO: 1 ) It increases binding to the priming receptor and/or reduces binding to the inhibitory receptor.

具有與CD32B降低的結合和/或與CD16A增加的結合的人IgG1 Fc區的示例性變異包含F243L、R292P、Y300L、V305I或P296L取代。這些氨基酸取代可以以任何組合存在於人IgG1 Fc區中。在一個實施方式中，變異的人IgG1 Fc區包含F243L、R292P和Y300L取代。在另一實施方式中，變異的人IgG1 Fc區包含F243L、R292P、Y300L、V305I和P296L取代。Exemplary variations of the human IgGl Fc region having reduced binding to CD32B and/or increased binding to CD16A comprise F243L, R292P, Y300L, V305I or P296L substitutions. These amino acid substitutions can be present in the human IgGl Fc region in any combination. In one embodiment, the variant human IgGl Fc region comprises F243L, R292P and Y300L substitutions. In another embodiment, the variant human IgGl Fc region comprises F243L, R292P, Y300L, V305I and P296L substitutions.

在某些實施方案中，本發明的ROR1-結合分子的Fc區優選地展示對FcγRIA (CD64)、FcγRIIA (CD32A)、FcγRIIB (CD32B)、FcγRIIIA (CD16a)或FcγRIIIB (CD16b)降低的(或基本上沒有)結合(相對於野生型IgG1 Fc區(SEQ ID NO: 1 )展示的結合)。在具體的實施方式中，本發明的包括IgG Fc區的ROR1-結合分子展示降低的ADCC效應子功能。在優選的實施方式中，這類ROR1-結合分子的CH2-CH3結構域包括下述的任何1、2、3或4個取代：L234A、L235A、D265A、N297Q和N297G。在另一實施方式中，CH2-CH3結構域包含N297Q取代、N297G取代、L234A和L235A替換或D265A取代，因為這些突變消除了FcR結合。可選地，使用固有地展示對FcγRIIIA (CD16a)降低的(或基本上沒有)結合和/或降低的效應子功能(相對於野生型IgG1 Fc區(SEQ ID NO: 1 )展示的結合和效應子功能)的天然產生的Fc區的CH2-CH3結構域。在具體的實施方式中，本發明的ROR1-結合分子包括IgG2 Fc區(SEQ ID NO:2 )或IgG4 Fc區(SEQ ID:NO:4 )。當使用IgG4 Fc區時，本發明也包括引入穩定化突變，比如上述的鉸鏈區S228P取代(見，例如，SEQ ID NO: 63 )。因為N297G、N297Q、L234A、L235A和D265A取代消除了效應子功能，在期望效應子功能的情況下，優選地將不採用這些取代。And X. There is no binding (binding shown relative to the wild type IgGl Fc region ( SEQ ID NO: 1 )). In a specific embodiment, a ROR1-binding molecule of the invention comprising an IgG Fc region exhibits reduced ADCC effector function. In a preferred embodiment, the CH2-CH3 domain of such ROR1-binding molecules includes any 1, 2, 3 or 4 substitutions as follows: L234A, L235A, D265A, N297Q and N297G. In another embodiment, the CH2-CH3 domain comprises a N297Q substitution, a N297G substitution, a L234A and L235A substitution, or a D265A substitution, as these mutations abolish FcR binding. Alternatively, the use of an effector function (in contrast to the wild-type IgGl Fc region ( SEQ ID NO: 1 ) exhibited by the FcγRIIIA (CD16a) reduced (or substantially no) binding and/or decreased expression is inherently displayed. Sub-function) of the CH2-CH3 domain of the naturally occurring Fc region. In a specific embodiment, the ROR1-binding molecule of the invention comprises an IgG2 Fc region ( SEQ ID NO: 2 ) or an IgG4 Fc region ( SEQ ID: NO: 4 ). When an IgG4 Fc region is used, the invention also encompasses the introduction of a stabilizing mutation, such as the hinge region S228P described above (see, for example, SEQ ID NO: 63 ). Since N297G, N297Q, L234A, L235A, and D265A substitutions eliminate effector functions, these substitutions would preferably not be employed where effector function is desired.

對於具有降低的或消除的效應子功能的本發明的包含Fc區的分子的CH2和CH3結構域，優選的IgG1序列包括取代L234A/L235A (SEQ ID NO:70 )： APE AA GGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG X 其中，X 為賴氨酸(K)或不存在。For the CH2 and CH3 domains of the Fc region-containing molecule of the invention having reduced or eliminated effector functions, the preferred IgG1 sequence comprises the substitution L234A/L235A ( SEQ ID NO: 70 ): APE AA GGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG X where X is lysine (K) or non-existent.

可通過增加Fc區對於FcRn的結合親和力而延長包括Fc區的蛋白質的血清半衰期。如本文使用的術語“半衰期”意思是分子的藥物代謝動力學性質，是對在其施用之後分子的平均生存時間的衡量。半衰期可表示為從受試者的身體(例如，人患者或其他哺乳動物)或其特定區室消除百分之五十(50%)的已知量的分子需要的時間，例如，如在血清中測量的，即，迴圈半衰期，或在其他組織中測量的。一般而言，半衰期的增加導致施用的分子在迴圈中的平均停留時間(MRT)的增加。The serum half-life of a protein including the Fc region can be extended by increasing the binding affinity of the Fc region for FcRn. The term "half-life" as used herein means the pharmacokinetic properties of a molecule, which is a measure of the average survival time of a molecule after its administration. The half-life can be expressed as the time required to eliminate fifty percent (50%) of a known amount of molecules from the subject's body (eg, a human patient or other mammal) or a particular compartment thereof, eg, as in serum Measured in, ie, half-life of the loop, or measured in other tissues. In general, an increase in half-life results in an increase in the mean residence time (MRT) of the administered molecule in the loop.

在一些實施方案中，本發明的ROR1-結合分子包括變異的Fc區，其中所述變異的Fc區相對於野生型Fc區包括至少一個氨基酸修飾，以便所述分子具有延長的半衰期(相對於包括野生型Fc區的分子)。在一些實施方式中，本發明的ROR1-結合分子包括變異的IgG Fc區，其中所述變異的Fc區在選自下述的一個或多個位置處包括延長半衰期的氨基酸取代：238、250、252、254、256、257、256、265、272、286、288、303、305、307、308、309、311、312、317、340、356、360、362、376、378、380、382、413、424、428、433、434、435和436。能夠延長包含Fc區的分子的半衰期的許多突變是本領域已知的，包括，例如M252Y、S254T、T256E和其組合。例如，見美國專利號6,277,375、7,083,784、7,217,797、8,088,376；美國公開號2002/0147311、2007/0148164；和PCT公開號WO 98/23289、WO 2009/058492和WO 2010/033279中描述的突變，其通過引用以它們的整體併入本文。具有延長的半衰期的ROR1-結合分子也包括具有在Fc區殘基250、252、254、256、257、288、307、308、309、311、378、428、433、434、435和436中的兩個或更多個處包括取代的變異的Fc區的那些。尤其地，兩個或更多個取代選自：T250Q、M252Y、S254T、T256E、K288D、T307Q、V308P、A378V、M428L、N434A、H435K和Y436I。In some embodiments, a ROR1-binding molecule of the invention comprises a variant Fc region, wherein the variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region such that the molecule has an extended half-life (relative to The molecule of the wild-type Fc region). In some embodiments, a ROR1-binding molecule of the invention comprises a variant IgG Fc region, wherein the variant Fc region comprises an amino acid substitution that extends half-life at one or more positions selected from the group consisting of: 238, 250, 252, 254, 256, 257, 256, 265, 272, 286, 288, 303, 305, 307, 308, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, 428, 433, 434, 435, and 436. Many mutations that are capable of extending the half-life of a molecule comprising an Fc region are known in the art and include, for example, M252Y, S254T, T256E, and combinations thereof. For example, see U.S. Patent Nos. 6,277,375, 7,083,784, 7,217,797, 8,088, 376, U.S. Publication Nos. 2002/0147311, 2007/0148164; and PCT Publication Nos. WO 98/23289, WO 2009/058492, and WO 2010/033279, which are incorporated by reference. The references are incorporated herein by reference in their entirety. ROR1-binding molecules having an extended half-life also include residues 250, 252, 254, 256, 257, 288, 307, 308, 309, 311, 378, 428, 433, 434, 435, and 436 in the Fc region. Two or more of those comprising a substituted variant Fc region. In particular, two or more substitutions are selected from the group consisting of: T250Q, M252Y, S254T, T256E, K288D, T307Q, V308P, A378V, M428L, N434A, H435K and Y436I.

在具體實施方案中，本發明的ROR1-結合分子具有包括以下取代的變異的IgG Fc區域： (A) M252Y、 S254T和T256E； (B) M252Y和S254T； (C) M252Y和T256E； (D) T250Q和M428L； (E) T307Q和N434A； (F) A378V和N434A； (G) N434A和Y436I； (H) V308P和N434A；或 (I) K288D和H435K。In a specific embodiment, the ROR1-binding molecule of the invention has an IgG Fc region comprising the following substituted variants: (A) M252Y, S254T and T256E; (B) M252Y and S254T; (C) M252Y and T256E; (D) T250Q and M428L; (E) T307Q and N434A; (F) A378V and N434A; (G) N434A and Y436I; (H) V308P and N434A; or (I) K288D and H435K.

在優選的實施方案中，本發明的ROR1-結合分子具有包括M252Y、S254T和T256E中的任何1、2或3個取代的變異的IgG Fc區域。本發明還包括具有變異的Fc區域的ROR1-結合分子，所述Fc區域包括： (A) 改變效應子功能和/或FcγR的一個或多個突變；和 (B) 延長血清半衰期的一個或多個突變。In a preferred embodiment, the ROR1-binding molecules of the invention have an IgG Fc region comprising any 1, 2 or 3 substituted variants of M252Y, S254T and T256E. The invention also encompasses ROR1-binding molecules having a variant Fc region comprising: (A) one or more mutations that alter effector function and/or FcγR; and (B) one or more of prolonging serum half-life A mutation.

對於其含有Fc區的第一和第三多肽鏈不一致的某些抗體、雙抗體和三價結合分子，期望減少或防止兩個第一多肽鏈的CH2-CH3結構域之間或兩個第三多肽鏈的CH2-CH3結構域之間發生同源二聚化。這類多肽鏈的CH2和/或CH3結構域不需要序列相同，並且有利地被修飾以促進兩條多肽鏈之間的複合。例如，氨基酸取代(優選以包括形成“杵(knob)”的大側基的氨基酸例如色氨酸進行取代)可被引入CH2或CH3結構域中，以便空間干擾將防止與類似的突變結構域的相互作用並將迫使突變的結構域與其中互補或適應性突變已經被工程化的結構域——即“臼(hole)”(例如，用甘氨酸取代)——配對。這樣的突變組可被工程化到任意對的、包括形成Fc區的CH2-CH3結構域的多肽中，以促進異源二聚化。蛋白質工程化以相對於同源二聚化利於異源二聚化的方法在本領域中是悉知的，尤其就工程化免疫球蛋白樣分子而言，這些都包括在本文中(見例如，Ridgway等(1996)“‘Knobs-Into-Holes’ Engineering Of Antibody CH3 Domains For Heavy Chain Heterodimerization,” Protein Engr. 9:617-621；Atwell等(1997)“Stable Heterodimers From Remodeling The Domain Interface Of A Homodimer Using A Phage Display Library,” J. Mol. Biol. 270: 26-35；和Xie等(2005)“A New Format Of Bispecific Antibody: Highly Efficient Heterodimerization, Expression And Tumor Cell Lysis,” J. Immunol. Methods 296:95-101；其每一篇通過引用以其整體併入本文)。For certain antibodies, diabodies and trivalent binding molecules that are inconsistent with the first and third polypeptide chains of the Fc region, it is desirable to reduce or prevent between the CH2-CH3 domains of the two first polypeptide chains or both Homologous dimerization occurs between the CH2-CH3 domains of the third polypeptide chain. The CH2 and/or CH3 domains of such polypeptide chains need not be identical in sequence and are advantageously modified to facilitate recombination between the two polypeptide chains. For example, an amino acid substitution (preferably substituted with an amino acid comprising a large side group that forms a "knob" such as tryptophan) can be introduced into the CH2 or CH3 domain such that steric interference will prevent similar mutated domains. The interaction will force the mutated domain to be paired with a domain in which the complementary or adaptive mutation has been engineered, ie, "hole" (eg, substituted with glycine). Such a set of mutations can be engineered into any pair of polypeptides comprising a CH2-CH3 domain that forms an Fc region to facilitate heterodimerization. Protein engineering is well known in the art as a method for heterodimerization relative to homodimerization, particularly in the context of engineered immunoglobulin-like molecules, as described herein (see, for example, Ridgway et al. (1996) "'Knobs-Into-Holes' Engineering Of Antibody CH3 Domains For Heavy Chain Heterodimerization," Protein Engr. 9:617-621; Atwell et al. (1997) "Stable Heterodimers From Remodeling The Domain Interface Of A Homodimer Using A Phage Display Library," J. Mol. Biol. 270: 26-35; and Xie et al. (2005) "A New Format Of Bispecific Antibody: Highly Efficient Heterodimerization, Expression And Tumor Cell Lysis," J. Immunol. Methods 296: 95-101; each of which is incorporated herein by reference in its entirety.

通過修飾IgG Fc區以包含修飾T366W而產生優選的杵。通過修飾IgG Fc區以包含修飾T366S、L368A和Y407V而產生優選的臼。為了有助於從最終的雙特異性的、異源二聚化的、包含Fc區的分子中純化含臼的第三多肽鏈同源二聚體，優選地通過在435位的氨基酸取代(H435R)突變第三多肽鏈的含臼的CH2和CH3結構域的蛋白質A結合位點。因此，含臼的第三多肽鏈同源二聚體不結合蛋白質A，而雙特異性的異源二聚體經在第一多肽鏈上的蛋白質A結合位點而保持其結合蛋白質A的能力。在可選的實施方式中，含臼的第三多肽鏈可併入在434和435位的氨基酸取代(N434A/N435K)。A preferred purine is produced by modifying the IgG Fc region to include the modified T366W. Preferred purines are produced by modifying the IgG Fc region to include modifications T366S, L368A and Y407V. To facilitate purification of the third polypeptide chain homodimer containing hydrazine from the final bispecific, heterodimerized, Fc region-containing molecule, preferably by amino acid substitution at position 435 ( H435R) mutates the protein A binding site of the purine-containing CH2 and CH3 domains of the third polypeptide chain. Thus, the third polypeptide-chain homodimer containing hydrazine does not bind to protein A, while the bispecific heterodimer retains its binding protein A via the protein A binding site on the first polypeptide chain. Ability. In an alternative embodiment, the third polypeptide chain containing ruthenium may incorporate amino acid substitutions at positions 434 and 435 (N434A/N435K).

對於本發明包含Fc區域的分子的第一多肽鏈的CH2和CH3結構域，優選的IgG氨基酸序列具有“包含杵的 ”序列(SEQ ID NO:71 )： APE AA GGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSL W CLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG X 其中，X 為賴氨酸(K)或不存在。For the CH2 and CH3 domains of the first polypeptide chain of the molecule comprising the Fc region of the invention, the preferred IgG amino acid sequence has a "purine- containing " sequence ( SEQ ID NO: 71 ): APE AA GGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSL W CLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG X where X is lysine (K) or non-existent.

對於具有兩條多肽鏈的本發明的包含Fc區域的分子的第二多肽鏈(或具有三、四或五條多肽鏈的包含Fc區域的分子的第三多肽鏈)的CH2和CH3結構域，優選的IgG氨基酸序列具有“包含臼的 ”序列(SEQ ID NO:72 )： APE AA GGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSL S C A VK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFL V SKL TVDKSRWQQG NVFSCSVMHE ALHN R YTQKS LSLSPG X 其中，X 為賴氨酸(K)或不存在。CH2 and CH3 domains of a second polypeptide chain of a Fc region-containing molecule of the invention having two polypeptide chains (or a third polypeptide chain of a molecule comprising an Fc region having three, four or five polypeptide chains) , preferably IgG amino acid sequence "comprises a hole" sequence (SEQ ID NO: 72): APE AA GGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSL S C a VK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFL V SKL TVDKSRWQQG NVFSCSVMHE ALHN R YTQKS LSLSPG X where X is lysine (K) or absent.

如將注意到的，SEQ ID NO:71 和SEQ ID NO:72 的CH2-CH3結構域包括在234用丙氨酸和235位用丙氨酸的取代，並且因此形成展示與FcγRIA (CD64)、FcγRIIA (CD32A)、FcγRIIB (CD32B)、FcγRIIIA (CD16a)或FcγRIIIB (CD16b)降低的(或基本上沒有)結合的Fc區(相對於野生型Fc區(SEQ ID NO:1 )展示的結合)。本發明也包括這類CH2-CH3結構域，其包括野生型丙氨酸殘基，修飾Fc區的效應子功能和/或FγR結合活性的可選的和/或另外取代。本發明也包括這類CH2-CH3結構域，其進一步包括一個或多個延長半衰期的氨基酸取代。尤其地，本發明包括這類包含臼的和這類包含杵的CH2-CH3結構域，其進一步包括M252Y/S254T/T256E。As will be noted, the CH2-CH3 domains of SEQ ID NO: 71 and SEQ ID NO: 72 include substitutions with alanine at position 234 and alanine at position 234, and thus form a display with FcyRIA (CD64), FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIIA (CD16a) or FcyRIIIB (CD16b) reduced (or substantially no) bound Fc region (binding as shown by the wild-type Fc region ( SEQ ID NO: 1 )). The invention also encompasses such CH2-CH3 domains, which include wild-type alanine residues, alternative and/or additional substitutions that modify the effector function and/or FyR binding activity of the Fc region. The invention also encompasses such CH2-CH3 domains, which further comprise one or more amino acid substitutions that extend half-life. In particular, the invention encompasses such CH2-CH3 domains comprising purines and such alfalfa, further comprising M252Y/S254T/T256E.

優選地，第一多肽鏈具有“包含杵的”CH2-CH3序列，比如SEQ ID NO:71 的序列。但是，如將認識到，“包含臼的”CH2-CH3結構域(例如，SEQ ID NO:72 )可用於第一多肽鏈中，在這類情況下，“包含杵的”CH2-CH3結構域(例如，SEQ ID NO:71 )用於本發明具有兩條多肽鏈的、包含Fc區的分子的第二多肽鏈中(或用於具有三、四或五條多肽鏈的包含Fc區的分子的第三多肽鏈中)。Preferably, the first polypeptide chain has a "purine-containing" CH2-CH3 sequence, such as the sequence of SEQ ID NO:71 . However, as will be appreciated, a "臼-containing" CH2-CH3 domain (eg, SEQ ID NO: 72 ) can be used in the first polypeptide chain, in which case the "杵-containing" CH2-CH3 structure A domain (eg, SEQ ID NO: 71 ) is used in a second polypeptide chain of a molecule comprising an Fc region of two polypeptide chains of the invention (or for an Fc region comprising three, four or five polypeptide chains) In the third polypeptide chain of the molecule).

在其他實施方案中，本發明包括ROR1-結合分子，其包括使用本領域已知的突變已經被工程化成相對於同源二聚化利於異源二聚化的CH2和/或CH3結構域，比如PCT公開號WO 2007/110205、WO 2011/143545、WO 2012/058768、WO 2013/06867中公開的那些，其所有通過引用以其整體併入本文。In other embodiments, the invention encompasses ROR1-binding molecules comprising CH2 and/or CH3 domains that have been engineered to facilitate heterodimerization relative to homodimerization using mutations known in the art, such as Those disclosed in PCT Publication Nos. WO 2007/110205, WO 2011/143545, WO 2012/058768, WO 2013/06867, all of which are incorporated herein in their entirety by reference.

VIII.VIII. 效應細胞結合能力Effector cell binding ability

如本文所提供的，本發明的ROR1-結合分子可以被工程化改造，以包括識別靶細胞或組織類型特有的一組抗原的表位元結合位元點的組合。尤其地，本發明涉及能夠結合ROR1的表位和存在於效應細胞(例如T淋巴細胞、自然殺傷(NK)細胞或其他單核細胞)表面上的分子的表位的多特異性ROR1-結合分子。例如，本發明的ROR1-結合分子可以構建成包括免疫特異性結合CD2、CD3、CD8、CD16、T細胞受體(TCR)或NKG2D的表位-結合位點。本發明還涉及能夠結合CD3的表位和CD8的表位的三特異性ROR1-結合分子(見例如PCT公開號WO 2015/026894)。As provided herein, the ROR1-binding molecules of the invention can be engineered to include a combination of epitope binding site points that recognize a panel of antigens specific to the target cell or tissue type. In particular, the present invention relates to a multispecific ROR1-binding molecule capable of binding to an epitope of ROR1 and an epitope present on a surface of an effector cell such as a T lymphocyte, natural killer (NK) cell or other monocyte. . For example, a ROR1-binding molecule of the invention can be constructed to include an epitope-binding site that immunospecifically binds to CD2, CD3, CD8, CD16, T cell receptor (TCR) or NKG2D. The invention also relates to a trispecific ROR1-binding molecule capable of binding to an epitope of CD3 and an epitope of CD8 (see, e.g., PCT Publication No. WO 2015/026894).

A. CD2A. CD2 結合能力Binding ability

在一個實施方案中，本發明的雙特異性、三特異性或多特異性ROR1-結合分子能夠結合ROR1的表位和CD2的表位。CD2是在T細胞和自然殺傷(NK)細胞的表面上發現的細胞粘附分子。CD2增強NK細胞細胞毒性，可能作為NK細胞納米管(nanotube)形成的啟動子(Mace, E.M.等(2014) “Cell Biological Steps and Checkpoints in Accessing NK Cell Cytotoxicity ,” Immunol. Cell. Biol. 92(3):245-255；Comerci, C.J.等(2012) “CD2 Promotes Human Natural Killer Cell Membrane Nanotube Formation ,” PLoS One 7(10):e47664:1-12)。特異性結合CD2的分子包括抗-CD2抗體“Lo-CD2a ”。In one embodiment, a bispecific, trispecific or multispecific ROR1-binding molecule of the invention is capable of binding to an epitope of ROR1 and an epitope of CD2. CD2 is a cell adhesion molecule found on the surface of T cells and natural killer (NK) cells. CD2 enhances NK cell cytotoxicity and may act as a promoter for NK cell nanotube formation (Mace, EM et al. (2014) " Cell Biological Steps and Checkpoints in Accessing NK Cell Cytotoxicity ," Immunol. Cell. Biol. 92 (3 ): 245-255; Comerci, CJ et al. (2012) " CD2 Promotes Human Natural Killer Cell Membrane Nanotube Formation ," PLoS One 7(10):e47664:1-12). Molecules that specifically bind to CD2 include the anti-CD2 antibody " Lo-CD2a ".

Lo-CD2a的VL結構域的氨基酸序列(ATCC登錄號：11423；SEQ ID NO:73 )如下所示(CDR_L 殘基以底線表示)： DVVLTQTPPT LLATIGQSVS ISC RSSQSLL HSSGNTYLN W LLQRTGQSPQ PLIY LVSKLE S GVPNRFSGS GSGTDFTLKI SGVEAEDLGV YYC MQFTHYP YT FGAGTKLE LKThe amino acid sequence of the VL domain of Lo-CD2a (ATCC Accession No.: 11423; SEQ ID NO: 73 ) is shown below (CDR _L residues are indicated by the bottom line): DVVLTQTPPT LLATIGQSVS ISC RSSQSLL HSSGNTYLN W LLQRTGQSPQ PLIY LVSKLE S GVPNRFSGS GSGTDFTLKI SGVEAEDLGV YYC MQFTHYP YT FGAGTKLE LK

Lo-CD2a的VH結構域的氨基酸序列(ATCC登錄號：11423；SEQ ID NO:74 )如下所示(CDR_H 殘基以底線表示)： EVQLQQSGPE LQRPGASVKL SCKASGYIFT EYYMY WVKQR PKQGLELVG R IDPEDGSIDY VEKFKK KATL TADTSSNTAY MQLSSLTSED TATYFCAR GK FNYRFAY WGQ GTLVTVSSThe amino acid sequence of the VH domain of Lo-CD2a (ATCC Accession No.: 11423; SEQ ID NO: 74 ) is shown below (CDR _H residues are indicated by the bottom line): EVQLQQSGPE LQRPGASVKL SCKASGYIFT EYYMY WVKQR PKQGLELVG R IDPEDGSIDY VEKFKK KATL TADTSSNTAY MQLSSLTSED TATYFCAR GK FNYRFAY WGQ GTLVTVSS

B. CD3B. CD3 結合能力Binding ability

在一個實施方案中，本發明的雙特異性、三特異性或多特異性ROR1-結合分子能夠結合ROR1的表位和CD3的表位。CD3是由四條不同的鏈組成的T-細胞共受體(Wucherpfennig, K.W.等(2010) “Structural Biology Of The T-Cell Receptor: Insights Into Receptor Assembly, Ligand Recognition, And Initiation Of Signaling ,” Cold Spring Harb. Perspect. Biol. 2(4):a005140; 第1-14頁)。在哺乳動物中，複合物包括CD3γ鏈、CD3δ鏈和兩條CD3ε鏈。這些鏈與稱為T-細胞受體(TCR)的分子締合，以在T淋巴細胞中產生啟動信號。在不存在CD3的情況下，TCR不能正確組裝並且被降解(Thomas, S.等(2010) “Molecular Immunology Lessons From Therapeutic T-Cell Receptor Gene Transfer ,” Immunology 129(2):170–177)。發現CD3結合於所有成熟T-細胞的膜，並且幾乎沒有其他細胞類型(參見，Janeway, C.A.等(2005), 在以下中: IMMUNOBIOLOGY: THE IMMUNE SYSTEM IN HEALTH AND DISEASE,” 第六版, Garland Science Publishing, NY, pp. 214- 216；Sun, Z. J.等(2001) “Mechanisms Contributing To T Cell Receptor Signaling And Assembly Revealed By The Solution Structure Of An Ectodomain Fragment Of The CD3ε:γ Heterodimer ,” Cell 105(7):913-923；Kuhns, M.S.等(2006) “Deconstructing The Form And Function Of The TCR/CD3 Complex ,” Immunity. 2006 Feb;24(2):133-139)。特異性結合CD3的分子包括抗-CD3抗體“CD3 mAb 1 ” 和“OKT3 ”。抗-CD3抗體CD3 mAb 1能夠結合非人靈長類動物(例如食蟹猴)。In one embodiment, a bispecific, trispecific or multispecific ROR1-binding molecule of the invention is capable of binding to an epitope of ROR1 and an epitope of CD3. CD3 is a T-cell co-receptor composed of four different chains (Wucherpfennig, KW et al. (2010) " Structural Biology Of The T-Cell Receptor : Insights Into Receptor Assembly, Ligand Recognition, And Initiation Of Signaling ," Cold Spring Harb Perspect. Biol. 2(4): a005140; pages 1-14). In mammals, the complex comprises a CD3 gamma chain, a CD3 delta chain and two CD3 epsilon chains. These chains associate with molecules called T-cell receptors (TCRs) to generate activating signals in T lymphocytes. In the absence of CD3, TCRs are not assembled correctly and are degraded (Thomas, S. et al. (2010) " Molecular Immunology Lessons From Therapeutic T-Cell Receptor Gene Transfer ," Immunology 129(2): 170-177). CD3 was found to bind to membranes of all mature T-cells and there were few other cell types (see, Janeway, CA et al. (2005), in the following: IMMUNOBIOLOGY: THE IMMUNE SYSTEM IN HEALTH AND DISEASE," Sixth Edition, Garland Science Publishing, NY, pp. 214-216; Sun, ZJ et al. (2001) “ Mechanisms Contributing To T Cell Receptor Signaling And Assembly Revealed By The Solution Structure Of An Ectodomain Fragment Of The CD3ε: γ Heterodimer ,” Cell 105(7): 913-923; Kuhns, MS et al. (2006) " Deconstructing The Form And Function Of The TCR/CD3 Complex ," Immunity. 2006 Feb; 24(2): 133-139). Molecules that specifically bind to CD3 include anti-CD3 The antibodies " CD3 mAb 1 " and " OKT3 ". The anti-CD3 antibody CD3 mAb 1 is capable of binding to non-human primates (eg, cynomolgus monkeys).

CD3 mAb 1的VL結構域的氨基酸序列(SEQ ID NO:75 )如下所示(CDR_L 殘基以底線表示)： QAVVTQEPSL TVSPGGTVTL TC RSSTGAVT TSNYAN WVQQ KPGQAPRGLI G GTNKRAP WT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWV F GGGTKLTVLGThe amino acid sequence of the VL domain of CD3 mAb 1 ( SEQ ID NO: 75 ) is shown below (CDR _L residues are indicated by the bottom line): QAVVTQEPSL TVSPGGTVTL TC RSSTGAVT TSNYAN WVQQ KPGQAPRGLI G GTNKRAP WT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWV F GGGTKLTVLG

CD3 mAb 1的VH結構域的氨基酸序列(SEQ ID NO:76 )如下所示(CDR_H 殘基以底線表示)： EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMN WVRQA PGKGLEWVG R IRSKYNNYAT YYADSVK D RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVS WFAY WGQGTL VTVSSThe amino acid sequence of the VH domain of CD3 mAb 1 ( SEQ ID NO: 76 ) is shown below (CDR _H residues are indicated by the bottom line): EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMN WVRQA PGKGLEWVG R IRSKYNNYAT YYADSVK D RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVS WFAY WGQGTL VTVSS

如下所述，為了闡釋本發明，製備了雙特異性ROR1 x CD3-結合分子。在一些ROR1 x CD3構建體中，使用CD3 mAb 1的變體。變異的“CD3 mAb 1(D65G) ”包括CD3 mAb 1的VL結構域(SEQ ID NO:75 )和具有D65G取代(Kabat位置65，對應於SEQ ID NO:77 的殘基68)的VH CD3 mAb 1結構域。To illustrate the invention, bispecific ROR1 x CD3-binding molecules were prepared as described below. In some ROR1 x CD3 constructs, variants of CD3 mAb 1 were used. The variant " CD3 mAb 1 (D65G) " includes the VL domain of CD3 mAb 1 ( SEQ ID NO: 75 ) and the VH CD3 mAb with D65G substitution (Kabat position 65, corresponding to residue 68 of SEQ ID NO: 77 ). 1 domain.

CD3 mAb 1(D65G)的VH的氨基酸序列(SEQ ID NO:77 )如下所示(CDR_H 殘基以底線表示，取代的位置(D65G)以雙底線表示)： EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMN WVRQA PGKGLEWVG R IRSKYNNYAT YYADSVK G RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVS WFAY WGQGTL VTVSSThe amino acid sequence of VH of CD3 mAb 1 (D65G) ( SEQ ID NO: 77 ) is shown below (CDR _H residues are indicated by the bottom line and the position of substitution (D65G) is indicated by the double bottom line): EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMN WVRQA PGKGLEWVG R IRSKYNNYAT YYADSVK G RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVS WFAY WGQGTL VTVSS

可選地，可以併入CD3 mAb 1的親和力變體。變體包括命名為“CD3 mAb 1 低 ”的低親和力變體和具有較快解離速率的命名為“CD3 mAb 1 快 ”的變體。CD mAb 1的VL結構域(SEQ ID NO:75 )對“CD3 mAb 1低”和“CD3 mAb 1快”來說是共有的，並在上文提供。下文提供了“CD3 mAb 1低”和“CD3 mAb 1快”各自的VH結構域的氨基酸序列。Alternatively, an affinity variant of CD3 mAb 1 can be incorporated. Variants include low affinity variants designated " CD3 mAb 1 low " and variants designated " CD3 mAb 1 fast " with faster dissociation rates. The VL domain of CD mAb 1 ( SEQ ID NO: 75 ) is common to "CD3 mAb 1 low" and "CD3 mAb 1 fast" and is provided above. The amino acid sequences of the respective VH domains of "CD3 mAb 1 low" and "CD3 mAb 1 fast" are provided below.

抗-人CD3 mAb 1低的可變重鏈結構域的氨基酸序列(SEQ ID NO:78 )如下所示(CDR_H 殘基以底線表示)： EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMN WVRQA PGKGLEWVG R IRSKYNNYAT YYADSVKG RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVT WFAY WGQGTL VTVSSThe amino acid sequence of the low variable heavy chain domain of anti-human CD3 mAb 1 ( SEQ ID NO: 78 ) is shown below (CDR _H residues are indicated by the bottom line): EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMN WVRQA PGKGLEWVG R IRSKYNNYAT YYADSVKG RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVT WFAY WGQGTL VTVSS

抗-人CD3 mAb 1快的可變重鏈結構域的氨基酸序列(SEQ ID NO:79 )如下所示(CDR_H 殘基以底線表示)： EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMN WVRQA PGKGLEWVG R IRSKYNNYAT YYADSVKG RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HKNFGNSYVT WFAY WGQGTL VTVSSThe amino acid sequence of the fast variable heavy chain domain of anti-human CD3 mAb 1 ( SEQ ID NO: 79 ) is shown below (CDR _H residues are indicated by the bottom line): EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMN WVRQA PGKGLEWVG R IRSKYNNYAT YYADSVKG RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HKNFGNSYVT WFAY WGQGTL VTVSS

可以使用的另一種抗CD3抗體是抗體莫羅單抗(Muromonab)-CD3“OKT3 ” (Xu等(2000)“In Vitro Characterization Of Five Humanized OKT3 Effector Function Variant Antibodies,” Cell. Immunol. 200:16-26)；Norman, D.J. (1995) “Mechanisms Of Action And Overview Of OKT3 ,” Ther. Drug Monit. 17(6):615-620；Canafax, D.M. 等(1987) “Monoclonal Antilymphocyte Antibody (OKT3) TreatmentOf Acute Renal Allograft Rejection ,” Pharmacotherapy 7(4):121-124；Swinnen, L.J.等(1993) “OKT3 Monoclonal Antibodies Induce Interleukin-6 And Interleukin-10: A Possible Cause Of Lymphoproliferative Disorders Associated With Transplantation ,” Curr. Opin. Nephrol. Hypertens. 2(4):670-678)。Another anti-CD3 antibody that can be used is the antibody muromonab-CD3 " OKT3 " (Xu et al. (2000) "In Vitro Characterization Of Five Humanized OKT3 Effector Function Variant Antibodies," Cell. Immunol. 200:16- 26); Norman, DJ (1995) “ Mechanisms Of Action And Overview Of OKT3 ,” Ther. Drug Monit. 17(6): 615-620; Canafax, DM et al. (1987) “ Monoclonal Antilymphocyte Antibody (OKT3) Treatment Of Acute Renal Allograft Rejection ," Pharmacotherapy 7(4): 121-124; Swinnen, LJ et al. (1993) " OKT3 Monoclonal Antibodies Induce Interleukin-6 And Interleukin-10: A Possible Cause Of Lymphoproliferative Disorders Associated With Transplantation ," Curr. Opin. Nephrol. Hypertens. 2(4): 670-678).

OKT3 的VL結構域的氨基酸序列(SEQ ID NO:80 )如下所示(CDR_L 殘基以底線表示)： QIVLTQSPAI MSASPGEKVT MTC SASSSVS YMN WYQQKSG TSPKRWIY DT SKLAS GVPAH FRGSGSGTSY SLTISGMEAE DAATYYC QQW SSNPFTF GSG TKLEINRThe amino acid sequence of VL domain of OKT3 ( SEQ ID NO: 80 ) is shown below (CDR _L residues are indicated by the bottom line): QIVLTQSPAI MSASPGEKVT MTC SASSSVS YMN WYQQKSG TSPKRWIY DT SKLAS GVPAH FRGSGSGTSY SLTISGMEAE DAATYYC QQW SSNPFTF GSG TKLEINR

O KT3 的VH結構域的氨基酸序列(SEQ ID NO:81 )如下所示(CDR_H 殘基以底線表示)： QVQLQQSGAE LARPGASVKM SCKASGYTFT RYTMH WVKQR PGQGLEWIG Y INPSRGYTNY NQKFKD KATL TTDKSSSTAY MQLSSLTSED SAVYYCAR YY DDHYCL DYWG QGTTLTVSSThe amino acid sequence of the VH domain of O KT3 ( SEQ ID NO: 81 ) is shown below (CDR _H residues are indicated by the bottom line): QVQLQQSGAE LARPGASVKM SCKASGYTFT RYTMH WVKQR PGQGLEWIG Y INPSRGYTNY NQKFKD KATL TTDKSSSTAY MQLSSLTSED SAVYYCAR YY DDHYCL DYWG QGTTLTVSS

可以被使用的另外的抗-CD3抗體包括但不限於在PCT公開號WO 2008/119566；和WO 2005/118635中所描述的那些。Additional anti-CD3 antibodies that can be used include, but are not limited to, those described in PCT Publication No. WO 2008/119566; and WO 2005/118635.

C. CD8C. CD8 結合能力Binding ability

在一個實施方案中，本發明的雙特異性、三特異性或多特異性ROR1-結合分子能夠結合ROR1的表位和CD8的表位。CD8是由兩條不同的鏈組成的T-細胞共受體(Leahy, D.J., (1995) “A Structural View of CD4 and CD8 ,” FASEB J., 9:17-25)，其在細胞毒性T-細胞上表達。已經發現CD8⁺ T-細胞的啟動是通過排列在靶細胞表面上的抗原：主要組織相容性I類(MHC I )分子複合物和排列在CD8⁺ T-細胞的表面上的CD8和T-細胞受體的複合物之間共刺激相互作用來介導的(Gao, G.,和Jakobsen, B., (2000). "Molecular interactions of coreceptor CD8 and MHC class I: the molecular basis for functional coordination with the T-Cell Receptor ". Immunol Today 21: 630–636)。與僅由某些免疫系統細胞表達的MHC II分子不同，MHC I分子被非常廣泛地表達。因此，細胞毒性T-細胞能夠結合多種細胞類型。啟動的細胞毒性T-細胞通過其釋放細胞毒素穿孔蛋白、粒酶和粒溶素而介導細胞殺傷。特異性結合CD8的抗體包括抗-CD8抗體“OKT8 ”和“TRX2 ”。In one embodiment, a bispecific, trispecific or multispecific ROR1-binding molecule of the invention is capable of binding to an epitope of ROR1 and an epitope of CD8. CD8 is a T-cell co-receptor composed of two different chains (Leahy, DJ, (1995) " A Structural View of CD4 and CD8 ," FASEB J., 9:17-25), which is in cytotoxic T - Expression on cells. It has been found that CD8 ⁺ T-cells are activated by antigens arranged on the surface of target cells: major histocompatibility class I ( MHC I ) molecular complexes and CD8 and T- arranged on the surface of CD8 ⁺ T-cells. Co-stimulatory interaction between cell receptor complexes is mediated (Gao, G., and Jakobsen, B., (2000). Molecular interactions of coreceptor CD8 and MHC class I: the molecular basis for functional coordination with The T-Cell Receptor ". Immunol Today 21: 630-636). Unlike MHC II molecules expressed only by certain immune system cells, MHC I molecules are expressed very broadly. Thus, cytotoxic T-cells are capable of binding to a variety of cell types. Activated cytotoxic T-cells mediate cell killing by releasing cytotoxic perforin, granzyme and granulysin. Antibodies that specifically bind to CD8 include the anti-CD8 antibodies " OKT8 " and " TRX2 ".

OKT8的VL結構域的氨基酸序列(SEQ ID NO:82 )如下所示(CDR_L 殘基以底線表示)： DIVMTQSPAS LAVSLGQRAT ISCRASESVD SYDNSLMH WY QQKPGQPPKV LIY LASNLES GVPARFSGSG SRTDFTLTID PVEADDAATY YC QQNNEDPY T FGGGTKLEI KRThe amino acid sequence of VL domain of OKT8 ( SEQ ID NO: 82 ) is shown below (CDR _L residues are indicated by the bottom line): DIVMTQSPAS LAVSLGQRAT ISCRASESVD SYDNSLMH WY QQKPGQPPKV LIY LASNLES GVPARFSGSG SRTDFTLTID PVEADDAATY YC QQNNEDPY T FGGGTKLEI KR

OKT8的VH結構域的氨基酸序列(SEQ ID NO:83)如下所示(CDR_H 殘基以底線表示)： QVQLLESGPE LLKPGASVKM SCKASGYTFT DYNMH WVKQS HGKSLEWIG Y IYPYTGGTGY NQKFKN KATL TVDSSSSTAY MELRSLTSED SAVYYCARNF RYTYWYFDVW GQGTTVTVSSThe amino acid sequence of the VH domain of OKT8 (SEQ ID NO: 83) is shown below (the CDR _H residue is indicated by the bottom line): QVQLLESGPE LLKPGASVKM SCKASGYTFT DYNMH WVKQS HGKSLEWIG Y IYPYTGGTGY NQKFKN KATL TVDSSSSTAY MELRSLTSED SAVYYCARNF RYTYWYFDVW GQGTTVTVSS

TRX2的VL結構域的氨基酸序列(SEQ ID NO:84)如下所示(CDR_L 殘基以底線表示)： DIQMTQSPSS LSASVGDRVT ITC KGSQDIN NYLA WYQQKP GKAPKLLIY N TDILHT GVPS RFSGSGSGTD FTFTISSLQP EDIATYYC YQ YNNGYT FGQG TKVEIKThe amino acid sequence of the VL domain of TRX2 (SEQ ID NO: 84) is shown below (CDR _L residues are indicated by the bottom line): DIQMTQSPSS LSASVGDRVT ITC KGSQDIN NYLA WYQQKP GKAPKLLIY N TDILHT GVPS RFSGSGSGTD FTFTISSLQP EDIATYYC YQ YNNGYT FGQG TKVEIK

TRX2的VH結構域的氨基酸序列(SEQ ID NO:85 )如下所示(CDR_H 殘基以底線表示)： QVQLVESGGG VVQPGRSLRL SCAASGFTFS DFGMN WVRQA PGKGLEWVA L IYYDGSNKFY ADSVKG RFTI SRDNSKNTLY LQMNSLRAED TAVYYCAK PH YDGYYHFFDS WGQGTLVTVS SThe amino acid sequence of the VH domain of TRX2 ( SEQ ID NO: 85 ) is shown below (CDR _H residues are indicated by the bottom line): QVQLVESGGG VVQPGRSLRL SCAASGFTFS DFGMN WVRQA PGKGLEWVA L IYYDGSNKFY ADSVKG RFTI SRDNSKNTLY LQMNSLRAED TAVYYCAK PH YDGYYHFFDS WGQGTLVTVS S

D. CD16D. CD16 結合能力Binding ability

在一個實施方案中，本發明的多特異性ROR1-結合分子能夠結合ROR1的表位和CD16的表位。CD16是FcγRIIIA受體。CD16由嗜中性粒細胞、嗜酸性粒細胞、自然殺傷(NK)細胞和結合聚集的的但不是單體的人IgG的組織巨噬細胞表達(Peltz, G.A.等(1989) “Human Fc Gamma RIII: Cloning, Expression, And Identification Of The Chromosomal Locus Of Two Fc Receptors For IgG ,” Proc. Natl. Acad. Sci. (U.S.A.) 86(3):1013-1017；Bachanova, V.等(2014) “NK Cells In Therapy Of Cancer ,” Crit. Rev. Oncog. 19(1-2):133-141；Miller, J.S. (2013) “Therapeutic Applications: Natural Killer Cells In The Clinic ,” Hematology Am. Soc. Hematol. Educ. Program. 2013:247-253；Youinou, P.等(2002) “Pathogenic Effects Of Anti-Fc Gamma Receptor IIIB (CD16) On Polymorphonuclear Neutrophils In Non-Organ-Specific Autoimmune Diseases ,” Autoimmun Rev. 1(1-2):13-19；Peipp, M.等(2002) “Bispecific Antibodies Targeting Cancer Cells ,” Biochem. Soc. Trans. 30(4):507-511)。特異性結合CD16的分子包括抗-CD16抗體“3G8 ”和“A9 ”。人源化的A9抗體在PCT公開號WO 03/101485中被描述。In one embodiment, a multispecific ROR1-binding molecule of the invention is capable of binding to an epitope of ROR1 and an epitope of CD16. CD16 is the FcγRIIIA receptor. CD16 is expressed by neutrophils, eosinophils, natural killer (NK) cells, and tissue macrophages that bind to aggregated but not monomeric human IgG (Peltz, GA et al. (1989) " Human Fc Gamma RIII : Cloning, Expression, And Identification Of The Chromosomal Locus Of Two Fc Receptors For IgG ,” Proc. Natl. Acad. Sci. (USA) 86(3):1013-1017; Bachanova, V. et al. (2014) “ NK Cells In Therapy Of Cancer ,” Crit. Rev. Oncog. 19(1-2): 133-141; Miller, JS (2013) “ Therapeutic Applications: Natural Killer Cells In The Clinic ,” Hematology Am. Soc. Hematol. Educ. Program. 2013: 247-253; Youinou, P. et al. (2002) “ Pathogenic Effects Of Anti-Fc Gamma Receptor IIIB (CD16) On Polymorphonuclear Neutrophils In Non-Organ-Specific Autoimmune Diseases ,” Autoimmun Rev. 1 (1-2 ): 13-19; Peipp, M. et al. (2002) " Bispecific Antibodies Targeting Cancer Cells ," Biochem. Soc. Trans. 30(4): 507-511). Molecules that specifically bind to CD16 include the anti-CD16 antibodies " 3G8 " and " A9 ". The humanized A9 antibody is described in PCT Publication No. WO 03/101485.

3G8的VL結構域的氨基酸序列(SEQ ID NO:86 )如下所示(CDR_L 殘基以底線表示)： DTVLTQSPAS LAVSLGQRAT ISC KASQSVD FDGDSFMN WY QQKPGQPPKL LIY TTSNLES GIPARFSASG SGTDFTLNIH PVEEEDTATY YC QQSNEDPY T FGGGTKLEI KThe amino acid sequence of VL domain of 3G8 ( SEQ ID NO: 86 ) is shown below (CDR _L residues are indicated by the bottom line): DTVLTQSPAS LAVSLGQRAT ISC KASQSVD FDGDSFMN WY QQKPGQPPKL LIY TTSNLES GIPARFSASG SGTDFTLNIH PVEEEDTATY YC QQSNEDPY T FGGGTKLEI K

3G8的VH結構域的氨基酸序列(SEQ ID NO:87)如下所示(CDR_H 殘基以底線表示)： QVTLKESGPG ILQPSQTLSL TCSFSGFSLR TSGMGVG WIR QPSGKGLEWL A HIWWDDDKR YNPALKS RLT ISKDTSSNQV FLKIASVDTA DTATYYCAQ I NPAWFAY WGQ GTLVTVSAThe amino acid sequence of the VH domain of 3G8 (SEQ ID NO: 87) is shown below (CDR _H residues are indicated by the bottom line): QVTLKESGPG ILQPSQTLSL TCSFSGFSLR TSGMGVG WIR QPSGKGLEWL A HIWWDDDKR YNPALKS RLT ISKDTSSNQV FLKIASVDTA DTATYYCAQ I NPAWFAY WGQ GTLVTVSA

A9的VL結構域的氨基酸序列(SEQ ID NO:88)如下所示(CDR_L 殘基以底線表示)： DIQAVVTQES ALTTSPGETV TLTC RSNTGT VTTSNYAN WV QEKPDHLFTG LIG HTNNRAP GVPARFSGSL IGDKAALTIT GAQTEDEAIY FC ALWYNNHW V FGGGTKLTVLThe amino acid sequence of VL domain of A9 (SEQ ID NO: 88) is shown below (CDR _L residues are indicated by the bottom line): DIQAVVTQES ALTTSPGETV TLTC RSNTGT VTTSNYAN WV QEKPDHLFTG LIG HTNNRAP GVPARFSGSL IGDKAALTIT GAQTEDEAIY FC ALWYNNHW V FGGGTKLTVL

A9的VH結構域的氨基酸序列(SEQ ID NO:89 )如下所示(CDR_H 殘基以底線表示)： QVQLQQSGAE LVRPGTSVKI SCKASGYTFT NYWLG WVKQR PGHGLEWIG D IYPGGGYTNY NEKFKG KATV TADTSSRTAY VQVRSLTSED SAVYFCAR SA SWYFD VWGAR TTVTVSSThe amino acid sequence of the VH domain of A9 ( SEQ ID NO: 89 ) is shown below (the CDR _H residue is indicated by the bottom line): QVQLQQSGAE LVRPGTSVKI SCKASGYTFT NYWLG WVKQR PGHGLEWIG D IYPGGGYTNY NEKFKG KATV TADTSSRTAY VQVRSLTSED SAVYFCAR SA SWYFD VWGAR TTVTVSS

可以使用的另外的抗-CD19抗體包括但不限於在PCT公開號WO 03/101485；和WO 2006/125668中描述的那些。Additional anti-CD19 antibodies that can be used include, but are not limited to, those described in PCT Publication No. WO 03/101485; and WO 2006/125668.

E. TCRE. TCR 結合能力Binding ability

在一個實施方案中，本發明的雙特異性、三特異性或多特異性ROR1-結合分子能夠結合ROR1的表位和T-細胞受體(TCR)的表位。T-細胞受體由CD4⁺ 或CD8⁺ T-細胞天然表達，並且允許這樣的細胞識別由抗原-呈遞細胞的I類或II類MHC蛋白結合和呈遞的抗原肽。通過TCR識別pMHC(肽–MHC)複合物啟動細胞免疫應答的傳播，其導致細胞因數的產生和抗原呈遞細胞的裂解(見例如，Armstrong, K.M.等(2008) “Conformational Changes And Flexibility In T-Cell Receptor Recognition Of Peptide–MHC Complexes ,” Biochem. J. 415(Pt 2):183–196；Willemsen, R. (2008) “Selection Of Human Antibody Fragments Directed Against Tumor T-Cell Epitopes For Adoptive T-Cell Therapy ,” Cytometry A. 73(11):1093-1099；Beier, K.C.等(2007) “Master Switches Of T-Cell Activation And Differentiation ,” Eur. Respir. J. 29:804-812；Mallone, R.等(2005) “Targeting T Lymphocytes For Immune Monitoring And Intervention In Autoimmune Diabetes ,” Am. J. Ther. 12(6):534–550)。CD3 是結合TCR的受體(Thomas, S.等(2010) “Molecular Immunology Lessons From Therapeutic T-Cell Receptor Gene Transfer ,” Immunology 129(2):170-177；Guy, C.S.等(2009) “Organization Of Proximal Signal Initiation At The TCR:CD3 Complex ,” Immunol. Rev. 232(1):7-21；St. Clair, E.W. (Epub 2009 Oct 12) “Novel Targeted Therapies For Autoimmunity ,” Curr. Opin. Immunol. 21(6):648-657；Baeuerle, P.A.等(Epub 2009 Jun 9) “Bispecific T-Cell Engaging Antibodies For Cancer Therapy ,” Cancer Res. 69(12):4941-4944；Smith-Garvin, J.E.等(2009) “T Cell Activation ,” Annu. Rev. Immunol. 27:591-619；Renders, L.等(2003) “Engineered CD3 Antibodies For Immunosuppression ,” Clin. Exp. Immunol. 133(3):307-309)。In one embodiment, a bispecific, trispecific or multispecific ROR1-binding molecule of the invention is capable of binding to an epitope of ROR1 and an epitope of a T-cell receptor (TCR). The T-cell receptor is naturally expressed by CD4 ⁺ or CD8 ⁺ T-cells and allows such cells to recognize antigenic peptides that are bound and presented by class I or class II MHC proteins of antigen-presenting cells. Recognition of the pMHC (peptide-MHC) complex by TCR initiates the spread of cellular immune responses leading to cytokine production and cleavage of antigen presenting cells (see, for example, Armstrong, KM et al. (2008) " Conformational Changes And Flexibility In T-Cell Receptor Recognition Of Peptide–MHC Complexes ,” Biochem. J. 415 (Pt 2): 183–196; Willemsen, R. (2008) “ Selection of Human Antibody Fragments Directed Against Tumor T-Cell Epitopes For Adoptive T-Cell Therapy , Cytometry A. 73(11): 1093-1099; Beier, KC et al. (2007) “ Master Switches Of T-Cell Activation And Differentiation ,” Eur. Respir. J. 29:804-812; Mallone, R. et al. 2005) " Targeting T Lymphocytes For Immune Monitoring And Intervention In Autoimmune Diabetes ," Am. J. Ther. 12(6): 534-550). CD3 is a receptor that binds to TCR (Thomas, S. et al. (2010) " Molecular Immunology Lessons From Therapeutic T-Cell Receptor Gene Transfer ," Immunology 129(2): 170-177; Guy, CS et al. (2009) " Organization Of Proximal Signal Initiation At The TCR: CD3 Complex ,” Immunol. Rev. 232(1):7-21; St. Clair, EW (Epub 2009 Oct 12) “ Novel Targeted Therapies For Autoimmunity ,” Curr. Opin. Immunol. 21 (6): 648-657; Baeuerle, PA et al. (Epub 2009 Jun 9) " Bispecific T-Cell Engaging Antibodies For Cancer Therapy ," Cancer Res. 69(12): 4941-4944; Smith-Garvin, JE et al (2009) " T Cell Activation ," Annu. Rev. Immunol. 27:591-619; Renders, L. et al. (2003) " Engineered CD3 Antibodies For Immunosuppression ," Clin. Exp. Immunol. 133(3): 307-309) .

特異性結合T-細胞受體的分子包括抗-TCR抗體“BMA 031 ” (EP 0403156；Kurrle, R.等(1989) “BMA 031 – A TCR-Specific Monoclonal Antibody For Clinical Application ,” Transplant Proc. 21(1 Pt 1):1017-1019；Nashan, B.等(1987) “Fine Specificity Of A Panel Of Antibodies Against The TCR/CD3 Complex, ” Transplant Proc. 19(5):4270-4272；Shearman, C.W.等(1991) “Construction, Expression, And Biologic Activity Of Murine/Human Chimeric Antibodies With Specificity For The Human α / β T Cell, ” J. Immunol. 146(3):928-935；Shearman, C.W. 等(1991) “Construction, Expression And Characterization of Humanized Antibodies Directed Against The Human α/β T Cell Receptor ,” J. Immunol. 147(12):4366-4373)。Molecules that specifically bind to T-cell receptors include the anti-TCR antibody " BMA 031 " (EP 0403156; Kurrle, R. et al. (1989) " BMA 031 - A TCR-Specific Monoclonal Antibody For Clinical Application ," Transplant Proc. 21 (1 Pt 1): 1017-1019; Nashan, B. et al. (1987) “ Fine Specificity Of A Panel Of Antibodies Against The TCR/CD3 Complex, ” Transplant Proc. 19(5): 4270-4272; Shearman, CW, etc. (1991) " Construction, Expression, And Biologic Activity Of Murine/Human Chimeric Antibodies With Specificity For The Human α / β T Cell, " J. Immunol. 146(3): 928-935; Shearman, CW et al. (1991) Construction, Expression And Characterization of Humanized Antibodies Directed Against The Human α/β T Cell Receptor ,” J. Immunol. 147(12): 4366-4373).

BMA 031的VL結構域的氨基酸序列(SEQ ID NO:90 )如下所示(CDR_L 殘基以底線表示)： EIVLTQSPAT LSLSPGERAT LSC SATSSVS YMH WYQQKPG KAPKRWIY DT SKLAS GVPSR FSGSGSGTEF TLTISSLQPE DFATYYC QQW SSNPLT FGQG TKLEIKThe amino acid sequence of the VL domain of BMA 031 ( SEQ ID NO: 90 ) is shown below (CDR _L residues are indicated by the bottom line): EIVLTQSPAT LSLSPGERAT LSC SATSSVS YMH WYQQKPG KAPKRWIY DT SKLAS GVPSR FSGSGSGTEF TLTISSLQPE DFATYYC QQW SSNPLT FGQG TKLEIK

BMA 031的VH結構域的氨基酸序列(SEQ ID NO:91 )如下所示(CDR_H 殘基以底線表示)： QVQLVQSGAE VKKPGASVKV SCKASGYKFT SYVMH WVRQA PGQGLEWIG Y INPYNDVTKY NEKFKG RVTI TADKSTSTAY LQMNSLRSED TAVHYCAR GS YYDYDGFVY W GQGTLVTVSSThe amino acid sequence of the VH domain of BMA 031 ( SEQ ID NO: 91 ) is shown below (CDR _H residues are indicated by the bottom line): QVQLVQSGAE VKKPGASVKV SCKASGYKFT SYVMH WVRQA PGQGLEWIG Y INPYNDVTKY NEKFKG RVTI TADKSTSTAY LQMNSLRSED TAVHYCAR GS YYDYDGFVY W GQGTLVTVSS

F. NKG2DF. NKG2D 結合能力Binding ability

在一個實施方案中，本發明的多特異性ROR1-結合分子能夠結合ROR1的表位和NKG2D受體的表位。NKG2D受體在所有人(和其他哺乳動物)的自然殺傷細胞上表達(Bauer, S.等(1999) “Activation Of NK Cells And T Cells By NKG2D, A Receptor For Stress-Inducible MICA ,” Science 285(5428):727-729；Jamieson, A.M.等(2002) “The Role Of The NKG2D Immunoreceptor In Immune Cell Activation And Natural Killing ,” Immunity 17(1):19-29)，也在所有CD8⁺ T細胞上表達(Groh, V.等(2001) “Costimulation Of CD8αβ T Cells By NKG2D Via Engagement By MIC Induced On Virus-Infected Cells ,” Nat. Immunol. 2(3):255-260；Jamieson, A.M.等(2002) “The Role Of The NKG2D Immunoreceptor In Immune Cell Activation And Natural Killing ,” Immunity 17(1):19-29)。這樣的結合配體，尤其是那些不在正常細胞上表達的結合配體，包括組織相容性60(H60)分子、視黃酸早期誘導基因-1(RAE-1)的產物和鼠UL16-結合蛋白樣轉錄物1 (MULT1) (Raulet D.H. (2003) “Roles Of The NKG2D Immunoreceptor And Its Ligands ,” Nature Rev. Immunol. 3:781-790；Coudert, J.D.等(2005)“Altered NKG2D Function In NK Cells Induced By Chronic Exposure To Altered NKG2D Ligand-Expressing Tumor Cells ,” Blood 106:1711-1717)。特異性結合NKG2D受體的分子包括抗-NKG2D抗體“KYK-1.0 ”和“KYK-2.0 ”(Kwong, KY等(2008) “Generation, Affinity Maturation, And Characterization Of A Human Anti-Human NKG2D Monoclonal Antibody With Dual Antagonistic And Agonistic Activity ,” J. Mol. Biol. 384:1143-1156；和PCT/US09/54911)。In one embodiment, the multispecific ROR1-binding molecules of the invention are capable of binding to an epitope of ROR1 and an epitope of the NKG2D receptor. NKG2D receptors are expressed on natural killer cells of all humans (and other mammals) (Bauer, S. et al. (1999) " Activation Of NK Cells And T Cells By NKG2D, A Receptor For Stress-Inducible MICA ," Science 285 ( 5428): 727-729; Jamieson, AM et al (2002) " The Role Of The NKG2D Immunoreceptor In Immune Cell Activation And Natural Killing ," Immunity 17(1): 19-29), also expressed on all CD8 ⁺ T cells (Groh, V. et al. (2001) " Costimulation Of CD8αβ T Cells By NKG2D Via Engagement By MIC Induced On Virus-Infected Cells ," Nat. Immunol. 2(3): 255-260; Jamieson, AM et al. (2002) The Role Of The NKG2D Immunoreceptor In Immune Cell Activation And Natural Killing ," Immunity 17(1): 19-29). Such binding ligands, especially those that are not expressed on normal cells, include histocompatibility 60 (H60) molecules, products of retinoic acid early induction gene-1 (RAE-1), and murine UL16-binding Protein-like transcript 1 (MULT1) (Raulet DH (2003) " Roles Of The NKG2D Immunoreceptor And Its Ligands ," Nature Rev. Immunol. 3:781-790; Coudert, JD et al. (2005) "Altered NKG2D Function In NK Cells Induced By Chronic Exposure To Altered NKG2D Ligand-Expressing Tumor Cells ," Blood 106: 1711-1717). Molecules that specifically bind to the NKG2D receptor include anti-NKG2D antibodies " KYK-1.0 " and " KYK-2.0 " (Kwong, KY et al. (2008) " Generation, Affinity Maturation, And Characterization Of A Human Anti-Human NKG2D Monoclonal Antibody With Dual Antagonistic And Agonistic Activity ," J. Mol. Biol. 384: 1143-1156; and PCT/US09/54911).

KYK-1.0的VL結構域的氨基酸序列(SEQ ID NO:92 )如下所示(CDR_L 殘基以底線表示)： QPVLTQPSSV SVAPGETARI PC GGDDIETK SVH WYQQKPG QAPVLVIY DD DDRPS GIPER FFGSNSGNTA TLSISRVEAG DEADYYC QVW DDNNDEWV FG GGTQLTVLThe amino acid sequence of the VL domain of KYK-1.0 ( SEQ ID NO: 92 ) is shown below (CDR _L residues are indicated by the bottom line): QPVLTQPSSV SVAPGETARI PC GGDDIETK SVH WYQQKPG QAPVLVIY DD DDRPS GIPER FFGSNSGNTA TLSISRVEAG DEADYYC QVW DDNNDEWV FG GGTQLTVL

KYK-1.0的VH結構域的氨基酸序列(SEQ ID NO:93)如下所示(CDR_H 殘基以底線表示)： EVQLVESGGG VVQPGGSLRL SCAASGFTFS SYGMH WVRQA PGKGLEWVA F IRYDGSNKYY ADSVKG RFTI SRDNSKNTKY LQMNSLRAED TAVYYCAK DR FGYYLDY WGQ GTLVTVSSThe amino acid sequence of the VH domain of KYK-1.0 (SEQ ID NO: 93) is shown below (CDR _H residues are indicated by the bottom line): EVQLVESGGG VVQPGGSLRL SCAASGFTFS SYGMH WVRQA PGKGLEWVA F IRYDGSNKYY ADSVKG RFTI SRDNSKNTKY LQMNSLRAED TAVYYCAK DR FGYYLDY WGQ GTLVTVSS

KYK-2.0的VL結構域的氨基酸序列(SEQ ID NO:94)如下所示(CDR_L 殘基以底線表示)： QSALTQPASV SGSPGQSITI SC SGSSSNIG NNAVN WYQQL PGKAPKLLIY YDDLLPS GVS DRFSGSKSGT SAFLAISGLQ SEDEADYYC A AWDDSLNGPV FGGGTKLTVLThe amino acid sequence of the VL domain of KYK-2.0 (SEQ ID NO: 94) is shown below (CDR _L residues are indicated by the bottom line): QSALTQPASV SGSPGQSITI SC SGSSSNIG NNAVN WYQQL PGKAPKLLIY YDDLLPS GVS DRFSGSKSGT SAFLAISGLQ SEDEADYYC A AWDDSLNGPV FGGGTKLTVL

KYK-2.0的VH結構域的氨基酸序列(SEQ ID NO:95 )如下所示(CDR_H 殘基以底線表示)： QVQLVESGGG LVKPGGSLRL SCAASGFTFS SYGMH WVRQA PGKGLEWVA F IRYDGSNKYY ADSVKG RFTI SRDNSKNTLY LQMNSLRAED TAVYYCAK DR GLGDGTYFDY WGQGTTVTVS SThe amino acid sequence of the VH domain of KYK-2.0 ( SEQ ID NO: 95 ) is shown below (CDR _H residues are indicated by the bottom line): QVQLVESGGG LVKPGGSLRL SCAASGFTFS SYGMH WVRQA PGKGLEWVA F IRYDGSNKYY ADSVKG RFTI SRDNSKNTLY LQMNSLRAED TAVYYCAK DR GLGDGTYFDY WGQGTTVTVS S

IXIX .. 示例性多特異性Exemplary multispecificity ROR1-ROR1- 結合分子Binding molecule

A. ROR1 x CD3A. ROR1 x CD3 雙特異性雙鏈雙抗體Bispecific double-stranded diabody

如本文所提供的，產生並表徵了三十三種示例性雙特異性雙鏈“ROR1 x CD3 ”雙抗體，其具有對ROR1特異性的一個結合位點(包括親本和/或優化的抗-ROR1-VL和抗-ROR1-VH結構域)和對CD3特異性的一個結合位點(包括CD3 mAb 1(D65G)的VL和VH結構域)。這樣的雙抗體被連續編號並命名為“DART-1 ”至“DART-33 ”。這些雙鏈雙特異性ROR1 x CD3雙抗體的結構在下面詳述。DART-1包括親本抗-ROR1-VL和抗-ROR1-VL結構域。這些示例性ROR1 x CD3雙特異性雙鏈雙抗體意圖是說明性的，但決不限制本發明的範圍。As provided herein, thirty-three exemplary bispecific double-stranded " ROR1 x CD3 " diabodyes are produced and characterized that have a binding site specific for ROR1 (including parental and/or optimized resistance) -ROR1-VL and anti-ROR1-VH domains) and a binding site specific for CD3 (including the VL and VH domains of CD3 mAb 1 (D65G)). Such diabody is serially numbered and designated " DART-1 " to " DART-33 ". The structure of these double-stranded bispecific ROR1 x CD3 diabody is detailed below. DART-1 includes the parental anti-ROR1-VL and anti-ROR1-VL domains. These exemplary ROR1 x CD3 bispecific duplex diabodies are intended to be illustrative, but in no way limit the scope of the invention.

示例性ROR1 x CD3雙特異性雙鏈雙抗體的第一多肽鏈在N-末端至C-末端方向包括：N-末端；選自S EQ ID NO:6 和SEQ ID NO:10-23 的抗-ROR1-VL結構域；間插間隔體肽(連接體1：GGGSGGGG (SEQ ID NO:33 ))；CD3 mAb 1(D65G)的VH結構域(SEQ ID NO:77 )；含有半胱氨酸的間插間隔體肽(連接體2：GGCGGG (SEQ ID NO:34 ))；異源二聚體促進(K-螺旋)結構域(KVAALKE-KVAALKE-KVAALKE-KVAALKE (SEQ ID NO:47 ))；和C-末端。每個雙抗體中存在的特定抗-ROR1-VL結構域顯示在表 7 中，並且氨基酸序列在上面提供。The first polypeptide chain of an exemplary ROR1 x CD3 bispecific duplex diabody comprises: an N-terminus in the N-terminal to C-terminal direction; and is selected from the group consisting of S EQ ID NO: 6 and SEQ ID NO: 10-23 anti-ROR1-VL domain; intervening spacer peptide (linker 1: GGGSGGGG ( SEQ ID NO: 33 )); VH domain of CD3 mAb 1 (D65G) ( SEQ ID NO: 77 ); containing cysteine Acid intervening spacer peptide (linker 2: GGCGGG ( SEQ ID NO: 34 )); heterodimer promoting (K-helix) domain (KVAALKE-KVAALKE-KVAALKE-KVAALKE ( SEQ ID NO: 47 ) ); and C-end. The specific anti-ROR1-VL domains present in each diabody are shown in Table 7 , and the amino acid sequences are provided above.

示例性ROR1 x CD3雙特異性雙鏈雙抗體的第二多肽鏈在N-末端至C-末端方向包括：N-末端；CD3 mAb 1的VL結構域(SEQ ID NO:75 )；間插間隔體肽(連接體1：GGGSGGGG (SEQ ID NO:33 ))；選自SEQ ID NO:7 和SEQ ID NO:24-32 的抗-ROR1-VH結構域；含有半胱氨酸的間插間隔體肽(連接體2：GGCGGG(SEQ ID NO:34 ))；異源二聚體促進(E-螺旋)結構域(EVAALEK-EVAALEK-EVAALEK-EVAALEK (SEQ ID NO:46 ))；和C-末端。每個雙抗體中存在的特定抗-ROR1-VH結構域顯示在表 7 中，並且氨基酸序列在上面提供。The second polypeptide chain of the exemplary ROR1 x CD3 bispecific double-stranded diabody comprises: N-terminal in the N-terminal to C-terminal direction; VL domain of CD3 mAb 1 ( SEQ ID NO: 75 ); Spacer peptide (linker 1: GGGSGGGG ( SEQ ID NO: 33 )); anti-ROR1-VH domain selected from SEQ ID NO: 7 and SEQ ID NO: 24-32 ; intervening containing cysteine Spacer peptide (linker 2: GGCGGG ( SEQ ID NO: 34 )); heterodimer-promoting (E-helical) domain (EVAALEK-EVAALEK-EVAALEK-EVAALEK ( SEQ ID NO: 46 )); - the end. The specific anti-ROR1-VH domain present in each diabody is shown in Table 7 , and the amino acid sequence is provided above.

DART-25DART-25

提供了代表性的ROR1 x CD3雙特異性雙鏈雙抗體DART-25的氨基酸序列。DART-25包括優化的抗-ROR1-VL結構域抗-ROR1-VL(2)和優化的抗-ROR1-VL結構域抗-ROR1-VL(7)。DART-25的CD3結合結構域是CD3 mAb 1(D65G)的VH結構域(SEQ ID NO:77 )和CD3 mAb 1的VL結構域(SEQ ID NO:75 )。抗-ROR1結合結構域和抗CD3結合結構域通過間插間隔體肽(連接體1：GGGSGGGG (SEQ ID NO:33 ))彼此分開。A representative amino acid sequence of the ROR1 x CD3 bispecific double-stranded diabody DART-25 is provided. DART-25 includes an optimized anti-ROR1-VL domain anti-ROR1-VL (2) and an optimized anti-ROR1-VL domain anti-ROR1-VL (7). The CD3 binding domain of DART-25 is the VH domain of CD3 mAb 1 (D65G) ( SEQ ID NO: 77 ) and the VL domain of CD3 mAb 1 ( SEQ ID NO: 75 ). The anti-ROR1 binding domain and the anti-CD3 binding domain are separated from each other by an intervening spacer peptide (linker 1: GGGSGGGG ( SEQ ID NO: 33 )).

DART-25的第一多肽鏈的氨基酸序列(SEQ ID NO:96 )如下所示(抗-ROR1-VL(2)以實底線表示；抗-CD3 mAb 1(D65G)的VH結構域以虛底線表示)： QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFGSGS SSGADWYLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLG GGGSGGG GEVQLVESGG GLVQPGGSLR LSCAASGFTF STYAMNWVRQ APGKGLEWVG RIRSKYNNYA TYYADSVKGR FTISRDDSKN SLYLQMNSLK TEDTAVYYCV RHGNFGNSYV SWFAYWGQGT LVTVSS GGCG GGKVAALKEK VAALKEKVAA LKEKVAALKEThe amino acid sequence of the first polypeptide chain of DART-25 ( SEQ ID NO: 96 ) is shown below (anti-ROR1-VL(2) is represented by the actual bottom line; the VH domain of anti-CD3 mAb 1 (D65G) is virtual The bottom line showing): QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFGSGS SSGADWYLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLG GGGSGGG G EVQLVESGG GLVQPGGSLR LSCAASGFTF STYAMNWVRQ APGKGLEWVG RIRSKYNNYA TYYADSVKGR FTISRDDSKN SLYLQMNSLK TEDTAVYYCV RHGNFGNSYV SWFAYWGQGT LVTVSS GGCG GGKVAALKEK VAALKEKVAA LKEKVAALKE

DART-25的第二多肽鏈的氨基酸序列(SEQ ID NO:97 )如下所示(抗-ROR1-VH(7)以實底線表示；CD3 mAb 1的VL結構域以虛底線表示)：QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGG QE QLVESGGGLV QPGGSLRLSC AASGFTFSDY YMSWVRQAPG KGLEWVATIY PSSGKTYYAD SVKGRLTISS DNAKDSLYLQ MNSLRAEDTA VYYCTRDSYA DDAALFDIWG QGTTVTVSS G GCGGGEVAAL EKEVAALEKE VAALEKEVAA LEKThe amino acid sequence of the second polypeptide chain of DART-25 ( SEQ ID NO: 97 ) is shown below (anti-ROR1-VH(7) is indicated by the solid underline; the VL domain of CD3 mAb 1 is indicated by the dashed bottom line): QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGG QE QLVESGGGLV QPGGSLRLSC AASGFTFSDY YMSWVRQAPG KGLEWVATIY PSSGKTYYAD SVKGRLTISS DNAKDSLYLQ MNSLRAEDTA VYYCTRDSYA DDAALFDIWG QGTTVTVSS G GCGGGEVAAL EKEVAALEKE VAALEKEVAA LEK

鑒於本文提供的教導，應當理解，可以利用不同的結構域取向、VH結構域、VL結構域、連接體和/或異源二聚體促進結構域來生成供選擇的ROR1 x CD3雙特異性雙鏈雙抗體。例如，使用不同的抗-ROR1-VL和/或VH結構域生成DART-1至DART-33(見例如表 7 )。此外，本文提供的優化的抗-ROR1-VL和/或VH結構域(優選SEQ ID NO:23 和SEQ ID NO:31 )中的任一個可以用於替代抗-ROR1-VL(2)和/或抗-ROR1-VH(7)，以生成供選擇的分子。In view of the teachings provided herein, it is to be understood that different domain orientations, VH domains, VL domains, linkers and/or heterodimer promoting domains can be utilized to generate alternative ROR1 x CD3 bispecific doubles. Chain diabody. For example, DART-1 to DART-33 are generated using different anti-ROR1-VL and/or VH domains (see, eg, Table 7 ). Furthermore, any of the optimized anti-ROR1-VL and/or VH domains provided herein (preferably SEQ ID NO: 23 and SEQ ID NO: 31 ) can be used in place of anti-ROR1-VL(2) and / Or anti-ROR1-VH(7) to generate alternative molecules.

B. ROR1 x CD3B. ROR1 x CD3 雙特異性三鏈雙抗體Bispecific triple chain diabody

如本文所提供的，產生並表徵四個示例性雙特異性三鏈“ROR1 x CD3 ”雙抗體，其具有對ROR1特異性的一個結合位點(包括親本和/或優化的抗-ROR1-VL和抗-ROR1-VH結構域)和對CD3特異性的一個結合位點(包括CD3 mAb 1(D65G)的VL和VH結構域)。示例性雙特異性三鏈雙抗體被命名如下：“DART-A ”，其包括親本的抗-ROR1-VL和抗-ROR1-VH結構域；“DART-B ”，其包括優化的抗-ROR1-VL(1)和親本的抗-ROR1-VH結構域； “DART-C ”，其包括優化的抗-ROR1-VL(14)和抗-ROR1-VH(7)結構域；和“DART-D ”，其包括優化的抗-ROR1-VL(14)和抗-ROR1-VH(8)結構域。這些ROR1 x CD3雙特異性三鏈雙抗體的結構在下面詳述。這些示例性ROR1 x CD3雙特異性三鏈雙抗體旨在闡釋，但決不限制本發明的範圍。As provided herein, four exemplary bispecific triplex " ROR1 x CD3 " diabodyes are produced and characterized, which have a binding site specific for ROR1 (including parental and/or optimized anti-ROR1- VL and anti-ROR1-VH domains) and a binding site specific for CD3 (including the VL and VH domains of CD3 mAb 1 (D65G)). An exemplary bispecific triplex diabody is named as follows: " DART-A ", which includes the anti-ROR1-VL and anti-ROR1-VH domains of the parent; " DART-B ", which includes optimized anti- ROR1-VL(1) and the anti-ROR1-VH domain of the parent; " DART-C ", which includes an optimized anti-ROR1-VL (14) and anti-ROR1-VH (7) domain; DART-D ", which includes optimized anti-ROR1-VL (14) and anti-ROR1-VH (8) domains. The structure of these ROR1 x CD3 bispecific triplex diabody is detailed below. These exemplary ROR1 x CD3 bispecific triplex diabody are intended to be illustrative, but in no way limit the scope of the invention.

示例性ROR1 x CD3雙特異性三鏈雙抗體的第一多肽鏈在N-末端至C-末端方向包括：N-末端；抗-ROR1-VL結構域(針對DART-A的S EQ ID NO:6 、針對DART-B的S EQ ID NO:10 、針對DART-C的S EQ ID NO:23 或針對DART-D的S EQ ID NO:23 )；間插間隔體肽(連接體1：GGGSGGGG (SEQ ID NO:33 ))；CD3 mAb 1(D65G)的VH結構域(SEQ ID NO:77 )；間插間隔體肽(連接體2：ASTKG(SEQ ID NO:38 ))；含有半胱氨酸的異源二聚體促進(E-螺旋)結構域(EVAACEK-EVAALEK-EVAALEK-EVAALEK (SEQ ID NO:48 ))；間插間隔體肽(連接體3：GGGDKTHTCPPCP (SEQ ID NO:58 ))；攜帶杵的IgG1 CH2-CH3結構域(SEQ ID NO:71 )；和C末端。該多肽鏈的編碼多核苷酸可以編碼SEQ ID NO:71 的C-末端賴氨酸殘基(即SEQ ID NO:71 的X )，然而，如上所述，該賴氨酸殘基在一些表達系統中可以被翻譯後去除。因此，本發明包括含有這樣的賴氨酸殘基的這樣的第一多肽鏈(即SEQ ID NO:71 ，其中X 為賴氨酸)，以及缺少這樣的賴氨酸殘基的第一多肽鏈(即SEQ ID NO:71 ，其中X 不存在)。每個雙抗體中存在的抗-ROR1-VL結構域顯示在表 9 中，並且氨基酸序列在下面提供。The first polypeptide chain of an exemplary ROR1 x CD3 bispecific triplex diabody comprises: an N-terminus in the N-terminal to C-terminal direction; an anti-ROR1-VL domain ( S EQ ID NO for DART-A) : 6 for DART-B of S EQ ID NO: 10, for DART-C of S EQ ID NO: 23 or for DART-D of S EQ ID NO: 23); intervening spacer peptide (linker 1: GGGSGGGG ( SEQ ID NO: 33 )); VH domain of CD3 mAb 1 (D65G) ( SEQ ID NO: 77 ); intervening spacer peptide (linker 2: ASTKG ( SEQ ID NO: 38 )); containing half Heterodimer-promoting (E-helical) domain of cystine (EVAACEK-EVAALEK-EVAALEK-EVAALEK ( SEQ ID NO: 48 )); intervening spacer peptide (linker 3: GGGDKTHTCPPCP ( SEQ ID NO: 58 )); carrying the IgG1 CH2-CH3 domain of sputum ( SEQ ID NO: 71 ); and the C-terminus. Encoding the polypeptide chain of the polynucleotide may encode SEQ ID NO: 71 C- terminal lysine residue (i.e., SEQ ID NO: 71 of the X-), however, as described above, the number of lysine residues in the expression The system can be removed after translation. Thus, the invention encompasses such a first polypeptide chain comprising lysine residues (ie SEQ ID NO: 71 wherein X is lysine), and the first number lacking such lysine residues Peptide chain (ie SEQ ID NO: 71 where X is absent). The anti-ROR1-VL domains present in each diabody are shown in Table 9 , and the amino acid sequences are provided below.

示例性ROR1 x CD3雙特異性三鏈雙抗體的第二多肽鏈在N-末端至C-末端方向包括：N-末端；CD3 mAb 1的VL結構域(SEQ ID NO:75 )；間插間隔體肽(連接體1：GGGSGGGG (SEQ ID NO:33 ))；抗-ROR1-VH結構域(針對DART-A的S EQ ID NO:7 、針對DART-B的S EQ ID NO:7 、針對DART-C的S EQ ID NO:30 、或針對DART-D的S EQ ID NO:31 )；間插間隔體肽(連接體2：ASTKG(SEQ ID NO:38 ))；含有半胱氨酸的異源二聚體促進(K-螺旋)結構域(KVAACKE-KVAALKE-KVAALKE-KVAALKE (SEQ ID NO:49 ))；和C-末端。每個雙抗體中存在的抗-ROR1-VH結構域顯示在表 9 中，並且氨基酸序列在下面提供。The second polypeptide chain of the exemplary ROR1 x CD3 bispecific triplex diabody comprises: the N-terminus in the N-terminal to C-terminal direction; the VL domain of CD3 mAb 1 ( SEQ ID NO: 75 ); spacer peptide (linker 1: GGGSGGGG (SEQ ID NO: 33)); anti -ROR1-VH domains (for DART-a of S EQ ID NO: 7, for the DART-B of S EQ ID NO: 7, S EQ ID NO: 30 for DART-C , or S EQ ID NO: 31 for DART-D; intervening spacer peptide (linker 2: ASTKG ( SEQ ID NO: 38 )); containing cysteine The heterodimer of the acid promotes the (K-helical) domain (KVAACKE-KVAALKE-KVAALKE-KVAALKE ( SEQ ID NO: 49 )); and the C-terminus. The anti-ROR1-VH domains present in each diabody are shown in Table 9 , and the amino acid sequences are provided below.

示例性ROR1 x CD3雙特異性三鏈雙抗體的第三多肽鏈在N-末端至C-末端方向包括：N-末端；間隔體肽(DKTHTCPPCP (SEQ ID NO:57 ))；攜帶臼的IgG1 CH2-CH3結構域(SEQ ID NO:72 )；和C-末端。該多肽鏈的編碼多核苷酸可以編碼SEQ ID NO:72 的C-末端賴氨酸殘基(即SEQ ID NO:72 的X )，然而，如上所述，該賴氨酸殘基在一些表達系統中可以被翻譯後去除。因此，本發明包括含這樣的類賴氨酸殘基的這樣的第三多肽鏈(即SEQ ID NO:72 ，其中X 為賴氨酸)，以及缺少這樣的賴氨酸殘基的第三多肽鏈(即SEQ ID NO:72 ，其中X 不存在)。第三多肽鏈對於每個示例性ROR1×CD3雙特異性三鏈雙抗體是共有的。A third polypeptide chain of an exemplary ROR1 x CD3 bispecific triplex diabody comprises: an N-terminus in the N-terminal to C-terminal direction; a spacer peptide (DKTHTCPPCP ( SEQ ID NO: 57 )); IgG1 CH2-CH3 domain ( SEQ ID NO: 72 ); and C-terminus. Encoding the polypeptide chain of the polynucleotide may encode SEQ ID NO: C- terminal lysine residue 72 (i.e., SEQ ID NO: X 72), whereas, as described above, the number of lysine residues in the expression The system can be removed after translation. Accordingly, the invention includes such a third polypeptide chain comprising such a lysine-like residue (ie, SEQ ID NO: 72 , wherein X is lysine), and a third lacking such a lysine residue Polypeptide chain (ie SEQ ID NO: 72 , wherein X is absent). The third polypeptide chain is common to each of the exemplary ROR1 x CD3 bispecific triplex diabody.

DART-ADART-A

因此，DART-A的第一多肽鏈的氨基酸序列(SEQ ID NO:98 )如下所示(親本抗-ROR1-VL用底線表示)： QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFGSGS SSGADRYLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLG GGGSGGG GEVQLVESGG GLVQPGGSLR LSCAASGFTF STYAMNWVRQ APGKGLEWVG RIRSKYNNYA TYYADSVKGR FTISRDDSKN SLYLQMNSLK TEDTAVYYCV RHGNFGNSYV SWFAYWGQGT LVTVSSASTK GEVAACEKEV AALEKEVAAL EKEVAALEKG GGDKTHTCPP CPAPEAAGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSREEM TKNQVSLWCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGKThus, the amino acid sequence of the first polypeptide chain of DART-A ( SEQ ID NO: 98 ) is shown below (parent anti-ROR1-VL is indicated by the bottom line): QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFGSGS SSGADRYLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLG GGGSGGG GEVQLVESGG GLVQPGGSLR LSCAASGFTF STYAMNWVRQ APGKGLEWVG RIRSKYNNYA TYYADSVKGR FTISRDDSKN SLYLQMNSLK TEDTAVYYCV RHGNFGNSYV SWFAYWGQGT LVTVSSASTK GEVAACEKEV AALEKEVAAL EKEVAALEKG GGDKTHTCPP CPAPEAAGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSREEM TKNQVSLWCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

DART-A的第二多肽鏈的氨基酸序列(SEQ ID NO:99 )如下所示(親本抗-ROR1-VH用底線表示)： QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGG QE QLVESGGGLV QPGGSLRLSC AASGFTFSDY YMSWVRQAPG KGLEWVATIY PSSGKTYYAD SVKGRFTISS DNAKNSLYLQ MNSLRAEDTA VYYCARDSYA DDAALFDIWG QGTTVTVSS A STKGKVAACK EKVAALKEKV AALKEKVAAL KEThe amino acid sequence of the second polypeptide chain of DART-A ( SEQ ID NO: 99 ) is shown below (parent anti-ROR1-VH is indicated by the bottom line): QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGG QE QLVESGGGLV QPGGSLRLSC AASGFTFSDY YMSWVRQAPG KGLEWVATIY PSSGKTYYAD SVKGRFTISS DNAKNSLYLQ MNSLRAEDTA VYYCARDSYA DDAALFDIWG QGTTVTVSS A STKGKVAACK EKVAALKEKV AALKEKVAAL KE

DART-A的第三多肽鏈的氨基酸序列為SEQ ID NO:100 ： DKTHTCPPCP APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLSCAVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLVSKL TVDKSRWQQG NVFSCSVMHE ALHNRYTQKS LSLSPGKDART-A amino acid sequence of the third polypeptide chain is SEQ ID NO: 100: DKTHTCPPCP APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLSCAVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLVSKL TVDKSRWQQG NVFSCSVMHE ALHNRYTQKS LSLSPGK

DART-BDART-B

DART-B的第一多肽鏈的氨基酸序列與DART-A的第一多肽鏈的氨基酸序列一致，除了Kabat位置63和64之間的G殘基缺失(以底線顯示)(SEQ ID NO:101 )： QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRF_ SGS SSGADRYLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLGGGGSGGG GEVQLVESGG GLVQPGGSLR LSCAASGFTF STYAMNWVRQ APGKGLEWVG RIRSKYNNYA TYYADSVKGR FTISRDDSKN SLYLQMNSLK TEDTAVYYCV RHGNFGNSYV SWFAYWGQGT LVTVSSASTK GEVAACEKEV AALEKEVAAL EKEVAALEKG GGDKTHTCPP CPAPEAAGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSREEM TKNQVSLWCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGKThe amino acid sequence of the first polypeptide chain of DART-B is identical to the amino acid sequence of the first polypeptide chain of DART-A, except for the deletion of the G residue between the Kabat positions 63 and 64 (shown by the bottom line) ( SEQ ID NO: 101): QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRF _ SGS SSGADRYLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLGGGGSGGG GEVQLVESGG GLVQPGGSLR LSCAASGFTF STYAMNWVRQ APGKGLEWVG RIRSKYNNYA TYYADSVKGR FTISRDDSKN SLYLQMNSLK TEDTAVYYCV RHGNFGNSYV SWFAYWGQGT LVTVSSASTK GEVAACEKEV AALEKEVAAL EKEVAALEKG GGDKTHTCPP CPAPEAAGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSREEM TKNQVSLWCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

DART-B的第二多肽鏈的氨基酸序列與DART-A的第二多肽鏈的氨基酸序列一致(SEQ ID NO:99 )。DART-B的第三多肽鏈的氨基酸序列與DART-A的第三多肽鏈的氨基酸序列一致(SEQ ID NO:100 )。The amino acid sequence of the second polypeptide chain of DART-B is identical to the amino acid sequence of the second polypeptide chain of DART-A ( SEQ ID NO: 99 ). The amino acid sequence of the third polypeptide chain of DART-B is identical to the amino acid sequence of the third polypeptide chain of DART-A ( SEQ ID NO: 100 ).

DART-CDART-C

DART-C的第一多肽鏈的氨基酸序列(SEQ ID NO:102 )如下所示(抗-ROR1-VL(14)用底線表示)： QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFSGSS SGADWYLTIS SLQSEDEADY YCGTDYPGNY LFGGGTQLTV LG GGGSGGGG EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMNWVRQA PGKGLEWVGR IRSKYNNYAT YYADSVKGRF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVS WFAYWGQGTL VTVSSASTKG EVAACEKEVA ALEKEVAALE KEVAALEKGG GDKTHTCPPC PAPEAAGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLWCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGKThe amino acid sequence of the first polypeptide chain of DART-C ( SEQ ID NO: 102 ) is shown below (anti-ROR1-VL (14) is indicated by the bottom line): QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFSGSS SGADWYLTIS SLQSEDEADY YCGTDYPGNY LFGGGTQLTV LG GGGSGGGG EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMNWVRQA PGKGLEWVGR IRSKYNNYAT YYADSVKGRF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVS WFAYWGQGTL VTVSSASTKG EVAACEKEVA ALEKEVAALE KEVAALEKGG GDKTHTCPPC PAPEAAGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLWCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK

DART-C的第二多肽鏈的氨基酸序列(SEQ ID NO:103 )如下所示(抗-ROR1-VH(7)用底線表示)： QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGG QE QLVESGGGLV QPGGSLRLSC AASGFTFSDY YMSWVRQAPG KGLEWVATIY PSSGKTYYAD SVKGRLTISS DNAKDSLYLQ MNSLRAEDTA VYYCTRDSYA DDAALFDIWG QGTTVTVSS A STKGKVAACK EKVAALKEKV AALKEKVAAL KEThe amino acid sequence of the second polypeptide chain of DART-C ( SEQ ID NO: 103 ) is shown below (anti-ROR1-VH(7) is indicated by the bottom line): QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGG QE QLVESGGGLV QPGGSLRLSC AASGFTFSDY YMSWVRQAPG KGLEWVATIY PSSGKTYYAD SVKGRLTISS DNAKDSLYLQ MNSLRAEDTA VYYCTRDSYA DDAALFDIWG QGTTVTVSS A STKGKVAACK EKVAALKEKV AALKEKVAAL KE

DART-C的第三多肽鏈的氨基酸序列與DART-A的第三多肽鏈的氨基酸序列一致(SEQ ID NO:100 )。The amino acid sequence of the third polypeptide chain of DART-C is identical to the amino acid sequence of the third polypeptide chain of DART-A ( SEQ ID NO: 100 ).

DART-DDART-D

DART-D的第一多肽鏈的氨基酸序列與DART-C的第一多肽鏈的氨基酸序列一致(SEQ ID NO:102 )。The amino acid sequence of the first polypeptide chain of DART-D is identical to the amino acid sequence of the first polypeptide chain of DART-C ( SEQ ID NO: 102 ).

DART-D的第二多肽鏈的氨基酸序列(SEQ ID NO:104 )如下所示(抗-ROR1-VH(8)用底線表示)： QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGG QE QLVESGGGLV QPGGSLRLSC AASGFTFSDY YMSWIRQAPG KGLEWVATIY PSSGKTYYAD SAKGRLTISS DNAKDSLYLQ MNSLRAEDTA VYYCTRDSYA DDAALFDIWG QGTTVTVSS A STKGKVAACK EKVAALKEKV AALKEKVAAL KEThe amino acid sequence of the second polypeptide chain of DART-D ( SEQ ID NO: 104 ) is shown below (anti-ROR1-VH(8) is indicated by the bottom line): QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGG QE QLVESGGGLV QPGGSLRLSC AASGFTFSDY YMSWIRQAPG KGLEWVATIY PSSGKTYYAD SAKGRLTISS DNAKDSLYLQ MNSLRAEDTA VYYCTRDSYA DDAALFDIWG QGTTVTVSS A STKGKVAACK EKVAALKEKV AALKEKVAAL KE

DART-D的第三多肽鏈的氨基酸序列與DART-A的第三多肽鏈的氨基酸序列一致(SEQ ID NO:100 )。The amino acid sequence of the third polypeptide chain of DART-D is identical to the amino acid sequence of the third polypeptide chain of DART-A ( SEQ ID NO: 100 ).

鑒於本文提供的教導，應當理解，可以利用不同的結構域取向、VH結構域、VL結構域、連接體、異源二聚體促進結構域和/或IgG恒定結構域來生成供選擇的ROR1 x CD3雙特異性三鏈雙抗體。例如，使用不同的抗-ROR1-VL和/或VH結構域生成DART-A至DART-D(見例如表 9 )。此外，本文提供的優化的抗-ROR1-VL和/或VH結構域中的任一個可以用於替代抗-ROR1-VL(14)和抗-ROR1-VH(8)，以生成供選擇的分子。In view of the teachings provided herein, it will be appreciated that different domain orientations, VH domains, VL domains, linkers, heterodimer promoting domains, and/or IgG constant domains can be utilized to generate alternative ROR1 x CD3 bispecific triplex diabody. For example, DART-A to DART-D are generated using different anti-ROR1-VL and/or VH domains (see, eg, Table 9 ). Furthermore, any of the optimized anti-ROR1-VL and/or VH domains provided herein can be used in place of anti-ROR1-VL (14) and anti-ROR1-VH (8) to generate alternative molecules. .

C. ROR1 x CD3 x CD8C. ROR1 x CD3 x CD8 三價結合分子Trivalent binding molecule

提供了示例性三價“ROR1 x CD3 x CD8 ”結合分子，其具有對ROR1特異性的一個結合位點(包括親本和/或優化的抗-ROR1-VL和抗-ROR1-VH結構域)、對CD3特異性的一個結合位點(包括CD3 mAb 1(D65G)的VL和VH結構域)和對CD8特異性的一個結合位點(包括TRX2的VL和VH結構域)。示例性三價結合分子被命名如下：“TRIDENT-A ”，其具有三條多肽鏈並且包括親本的抗-ROR1-VL和抗-ROR1-VH結構域；“TRIDENT-B ” ，其具有四條多肽鏈並且包括親本的抗-ROR1-VL和抗-ROR1-VH結構域；“TRIDENT-C ” ，其具有三條多肽鏈並且包括優化的抗-ROR1-VL(14)和抗-ROR1-VH(8)結構域； “TRIDENT-D ” ，其具有四條多肽鏈並且包括優化的抗-ROR1-VL(14)和抗-ROR1-VH(8)結構域。TRIDENT-A和TRIDENT-C具有圖 6D 顯示的一般結構，並且TRIDENT-B和TRIDENT-D具有圖 6A 顯示的一般結構。這些ROR1 x CD3 x CD8三價結合分子的結構在下面詳述。這些示例性ROR1 x CD3 x CD8三價結合分子旨在闡釋，但決不限制本發明的範圍。An exemplary trivalent " ROR1 x CD3 x CD8 " binding molecule is provided which has a binding site specific for ROR1 (including parental and/or optimized anti-ROR1-VL and anti-ROR1-VH domains) A binding site specific for CD3 (including the VL and VH domains of CD3 mAb 1 (D65G)) and a binding site specific for CD8 (including the VL and VH domains of TRX2). An exemplary trivalent binding molecule is named as follows: " TRIDENT-A ", which has three polypeptide chains and includes the parental anti-ROR1-VL and anti-ROR1-VH domains; " TRIDENT-B " with four polypeptides The chain also includes the parental anti-ROR1-VL and anti-ROR1-VH domains; " TRIDENT-C ", which has three polypeptide chains and includes optimized anti-ROR1-VL (14) and anti-ROR1-VH ( 8) Domain; " TRIDENT-D ", which has four polypeptide chains and includes optimized anti-ROR1-VL (14) and anti-ROR1-VH (8) domains. TRIDENT-A and TRIDENT-C have the general structure shown in Figure 6D , and TRIDENT-B and TRIDENT-D have the general structure shown in Figure 6A . The structure of these ROR1 x CD3 x CD8 trivalent binding molecules is detailed below. These exemplary ROR1 x CD3 x CD8 trivalent binding molecules are intended to be illustrative, but in no way limit the scope of the invention.

具有三條或四條多肽鏈的示例性ROR1 x CD3 x CD8三價結合分子(見例如，圖 6A )的第一多肽鏈在N-末端至C-末端方向包括：N-末端；抗-ROR1-VL結構域(針對TRIDENT-A的S EQ ID NO:6 、針對TRIDENT-B的S EQ ID NO:6 、針對TRIDENT-C的S EQ ID NO:23 和針對TRIDENT-D的S EQ ID NO:23 )；間插間隔體肽(連接體1：GGGSGGGG (SEQ ID NO:33 ))；CD3 mAb 1(D65G)的VH結構域(SEQ ID NO:77 )；間插間隔體肽(連接體2：ASTKG(SEQ ID NO:38 ))；含有半胱氨酸的異源二聚體促進(E-螺旋)結構域(EVAACEK-EVAALEK-EVAALEK-EVAALEK (SEQ ID NO:48 ))；間插間隔體肽(連接體3：GGGDKTHTCPPCP (SEQ ID NO:58 ))；攜帶杵的IgG1 CH2-CH3結構域(SEQ ID NO:71 )；和C末端。該多肽鏈的編碼多核苷酸可以編碼SEQ ID NO:71 的C-末端賴氨酸殘基(即SEQ ID NO:71 的X )，然而，如上所述，該賴氨酸殘基在一些表達系統中可以被翻譯後去除。因此，本發明包括含有這樣的賴氨酸殘基的這樣的第一多肽鏈(即SEQ ID NO:71 ，其中X 為賴氨酸)，以及缺少這樣的賴氨酸殘基的第一多肽鏈(即SEQ ID NO:71 ，其中X 不存在)。每個三價結合分子中存在的抗-ROR1-VL結構域顯示在表 10 中，並且氨基酸序列在上面提供。The first polypeptide chain of an exemplary ROR1 x CD3 x CD8 trivalent binding molecule having three or four polypeptide chains (see, eg, Figure 6A ) comprises: N-terminal in the N-terminal to C-terminal direction; anti-ROR1- VL domain (for TRIDENT-a of S EQ ID NO: 6, for TRIDENT-B of S EQ ID NO: 6, for TRIDENT-C of S EQ ID NO: 23 and for TRIDENT-D of S EQ ID NO: 23 ); intervening spacer peptide (linker 1: GGGSGGGG ( SEQ ID NO: 33 )); VH domain of CD3 mAb 1 (D65G) ( SEQ ID NO: 77 ); intervening spacer peptide (linker 2) : ASTKG ( SEQ ID NO: 38 )); a heterodimer-promoting (E-helical) domain containing cysteine (EVAACEK-EVAALEK-EVAALEK-EVAALEK ( SEQ ID NO: 48 )); intervening spacer The peptide (linker 3: GGGDKTHTCPPCP ( SEQ ID NO: 58 )); the IgG1 CH2-CH3 domain carrying the sputum ( SEQ ID NO: 71 ); and the C-terminus. Encoding the polypeptide chain of the polynucleotide may encode SEQ ID NO: 71 C- terminal lysine residue (i.e., SEQ ID NO: 71 of the X-), however, as described above, the number of lysine residues in the expression The system can be removed after translation. Thus, the invention encompasses such a first polypeptide chain comprising lysine residues (ie SEQ ID NO: 71 wherein X is lysine), and the first number lacking such lysine residues Peptide chain (ie SEQ ID NO: 71 where X is absent). The anti-ROR1-VL domain present in each trivalent binding molecule is shown in Table 10 , and the amino acid sequence is provided above.

具有三條或四條多肽鏈的示例性ROR1 x CD3 x CD8三價結合分子的第二多肽鏈在N-末端至C-末端方向包括：N-末端；CD3 mAb 1的VL結構域(SEQ ID NO:75 )；間插間隔體肽(連接體1：GGGSGGGG (SEQ ID NO:33 ))；抗-ROR1-VH結構域(針對TRIDENT-A的S EQ ID NO:7 、針對TRIDENT-B的S EQ ID NO:7 、針對TRIDENT-C的S EQ ID NO:31 、或針對TRIDENT-D的S EQ ID NO:31 )；間插間隔體肽(連接體2：ASTKG(SEQ ID NO:38 ))；含有半胱氨酸的異源二聚體促進(K-螺旋)結構域(KVAACKE-KVAALKE-KVAALKE-KVAALKE (SEQ ID NO:49 ))；和C末端。每個雙抗體中存在的抗-ROR1-VH結構域顯示在表 10 中，並且氨基酸序列在上面提供。A second polypeptide chain of an exemplary ROR1 x CD3 x CD8 trivalent binding molecule having three or four polypeptide chains includes: an N-terminus in the N-terminal to C-terminal direction; a VL domain of CD3 mAb 1 ( SEQ ID NO : 75); intervening spacer peptide (linker 1: GGGSGGGG (SEQ ID NO: 33)); anti -ROR1-VH domains (for TRIDENT-a of S EQ ID NO: 7, for the S TRIDENT-B EQ ID NO: 7, for TRIDENT-C of S EQ ID NO: 31, or for TRIDENT-D of S EQ ID NO: 31); intervening spacer peptide (linker 2: ASTKG (SEQ ID NO: 38) a cysteine-containing heterodimer-promoting (K-helical) domain (KVAACKE-KVAALKE-KVAALKE-KVAALKE ( SEQ ID NO: 49 )); and a C-terminus. The anti-ROR1-VH domains present in each diabody are shown in Table 10 , and the amino acid sequence is provided above.

示例性三多肽鏈ROR1 x CD3 x CD8三價結合分子TRIDENT-A和TRIDENT-C的第三多肽鏈在N-末端至C-末端方向包括：N-末端；TRX2的VL結構域(SEQ ID NO:84 )；間插間隔體肽(連接體4：GGGGSGGGGSGGGGS (SEQ ID NO:64 ))；TRX2的VH結構域(SEQ ID NO:85 )；間插間隔體肽(連接體3：VEPKSADKTHTCPPCP (SEQ ID NO:55 ))；攜帶臼的IgG1 CH2-CH3結構域(SEQ ID NO:72 )；和C末端。該多肽鏈的編碼多核苷酸可以編碼SEQ ID NO:72 的C-末端賴氨酸殘基(即，SEQ ID NO:72 的X )，然而，如上所述，該賴氨酸殘基在一些表達系統中可以被翻譯後去除。因此，本發明包括含有這樣的賴氨酸殘基的這樣的第三多肽鏈(即SEQ ID NO:72 ，其中X 為賴氨酸)，以及缺少這樣的賴氨酸殘基的第三多肽鏈(即SEQ ID NO:72 ，其中X 不存在)。Exemplary third polypeptide chain ROR1 x CD3 x CD8 trivalent binding molecules The third polypeptide chain of TRIDENT-A and TRIDENT-C includes: N-terminal in the N-terminal to C-terminal direction; VL domain of TRX2 ( SEQ ID NO: 84 ); intervening spacer peptide (linker 4: GGGGSGGGGSGGGGS ( SEQ ID NO: 64 )); VH domain of TRX2 ( SEQ ID NO: 85 ); intervening spacer peptide (linker 3: VEPKSADKTHTCPPCP ( SEQ ID NO: 55 )); an IgG1 CH2-CH3 domain carrying the sputum ( SEQ ID NO: 72 ); and the C-terminus. Encoding the polypeptide chain of the polynucleotide may encode SEQ ID NO: C- terminal lysine residue 72 (i.e., SEQ ID NO: X 72), whereas, as mentioned above, some of the lysine residue The expression system can be removed after translation. Thus, the invention encompasses such a third polypeptide chain comprising lysine residues (i.e., SEQ ID NO: 72 , wherein X is lysine), and the third most lacking such lysine residues Peptide chain (ie SEQ ID NO: 72 , wherein X is absent).

示例性四多肽鏈ROR1 x CD3 x CD8三價結合分子TRIDENT-B和TRIDENT-D的第三多肽鏈是抗體重鏈，其在N-末端至C-末端方向包括：N-末端；TRX2的VH結構域(SEQ ID NO:85 )；IgG1 CH1結構域(SEQ ID NO:67 )；IgG1鉸鏈區域(EPKSCDKTHTCPPCP(SEQ ID NO:60 ))；攜帶臼的IgG1 CH2-CH3結構域(SEQ ID NO:72 )；和C末端。該多肽鏈的編碼多核苷酸可以編碼SEQ ID NO:72 的C-末端賴氨酸殘基(即SEQ ID NO:72 的X )，然而，如上所述，該賴氨酸殘基在一些表達系統中可以被翻譯後去除。因此，本發明包括含有這樣的賴氨酸殘基的這樣的第三多肽鏈(即SEQ ID NO:72 ，其中X 為賴氨酸)，以及缺少這樣的賴氨酸殘基的第三多肽鏈(即SEQ ID NO:72 ，其中X 不存在)。Exemplary fourth polypeptide chain ROR1 x CD3 x CD8 trivalent binding molecules The third polypeptide chain of TRIDENT-B and TRIDENT-D is an antibody heavy chain comprising: N-terminal in the N-terminal to C-terminal direction; TRX2 VH domain ( SEQ ID NO:85 ); IgG1 CH1 domain ( SEQ ID NO:67 ); IgG1 hinge region (EPKSCDKTHTCPPCP ( SEQ ID NO:60 )); 臼1 carrying IgG1 CH2-CH3 domain ( SEQ ID NO: 72 ); and C-terminal. Encoding the polypeptide chain of the polynucleotide may encode SEQ ID NO: C- terminal lysine residue 72 (i.e., SEQ ID NO: X 72), whereas, as described above, the number of lysine residues in the expression The system can be removed after translation. Thus, the invention encompasses such a third polypeptide chain comprising lysine residues (i.e., SEQ ID NO: 72 , wherein X is lysine), and the third most lacking such lysine residues Peptide chain (ie SEQ ID NO: 72 , wherein X is absent).

具有四條多肽鏈的示例性ROR1 x CD3 x CD8三價結合分子(即，TRIDENT-B和TRIDENT-D)的第四多肽鏈是抗體輕鏈，其在N-末端至C-末端方向包括：N-末端；CD8 mAb TRX2的VL結構域(SEQ ID NO:84 )；CL κ結構域(SEQ ID NO:65 )；和C末端。A fourth polypeptide chain of an exemplary ROR1 x CD3 x CD8 trivalent binding molecule having four polypeptide chains (ie, TRIDENT-B and TRIDENT-D) is an antibody light chain comprising, in the N-terminal to C-terminal orientation: N-terminus; VL domain of CD8 mAb TRX2 ( SEQ ID NO: 84 ); CL κ domain ( SEQ ID NO: 65 ); and C-terminus.

提供了具有三條多肽鏈的代表性的ROR1 x CD3 x CD8三價結合分子TRIDENT-C的氨基酸序列。TRIDENT-C分別包括優化的抗-ROR1-VL和抗-ROR1-VH結構域抗-ROR1-VL(14)和抗-ROR1-VH(8)。The amino acid sequence of a representative ROR1 x CD3 x CD8 trivalent binding molecule TRIDENT-C having three polypeptide chains is provided. TRIDENT-C includes optimized anti-ROR1-VL and anti-ROR1-VH domain anti-ROR1-VL (14) and anti-ROR1-VH (8), respectively.

TRIDENT-ATRIDENT-A

TRIDENT-A的第一多肽鏈的氨基酸序列(SEQ ID NO:105 )如下所示(親本抗-ROR1-VL以底線表示)： QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFGSGS SSGADRYLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLG GGGSGGG GEVQLVESGG GLVQPGGSLR LSCAASGFTF STYAMNWVRQ APGKGLEWVG RIRSKYNNYA TYYADSVKGR FTISRDDSKN SLYLQMNSLK TEDTAVYYCV RHGNFGNSYV SWFAYWGQGT LVTVSSASTK GEVAACEKEV AALEKEVAAL EKEVAALEKG GGDKTHTCPP CPAPEAAGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSREEM TKNQVSLWCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGKThe amino acid sequence of the first polypeptide chain of TRIDENT-A ( SEQ ID NO: 105 ) is shown below (parent anti-ROR1-VL is indicated by the bottom line): QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFGSGS SSGADRYLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLG GGGSGGG GEVQLVESGG GLVQPGGSLR LSCAASGFTF STYAMNWVRQ APGKGLEWVG RIRSKYNNYA TYYADSVKGR FTISRDDSKN SLYLQMNSLK TEDTAVYYCV RHGNFGNSYV SWFAYWGQGT LVTVSSASTK GEVAACEKEV AALEKEVAAL EKEVAALEKG GGDKTHTCPP CPAPEAAGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSREEM TKNQVSLWCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

TRIDENT-A的第二多肽鏈的氨基酸序列(SEQ ID NO:106 )如下所示(親本抗-ROR1-VH以底線表示)： QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGG QE QLVESGGGLV QPGGSLRLSC AASGFTFSDY YMSWVRQAPG KGLEWVATIY PSSGKTYYAD SVKGRFTISS DNAKNSLYLQ MNSLRAEDTA VYYCARDSYA DDAALFDIWG QGTTVTVSS A STKGKVAACK EKVAALKEKV AALKEKVAAL KEThe amino acid sequence of the second polypeptide chain of TRIDENT-A ( SEQ ID NO: 106 ) is shown below (parent anti-ROR1-VH is indicated by the bottom line): QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGG QE QLVESGGGLV QPGGSLRLSC AASGFTFSDY YMSWVRQAPG KGLEWVATIY PSSGKTYYAD SVKGRFTISS DNAKNSLYLQ MNSLRAEDTA VYYCARDSYA DDAALFDIWG QGTTVTVSS A STKGKVAACK EKVAALKEKV AALKEKVAAL KE

TRIDENT-A的第三多肽鏈的氨基酸序列(SEQ ID NO:107 )如下所示： DIQMTQSPSS LSASVGDRVT ITCKGSQDIN NYLAWYQQKP GKAPKLLIYN TDILHTGVPS RFSGSGSGTD FTFTISSLQP EDIATYYCYQ YNNGYTFGCG TKVEIKGGGG SGGGGSGGGG SQVQLVESGG GVVQPGRSLR LSCAASGFTF SDFGMNWVRQ APGKCLEWVA LIYYDGSNKF YADSVKGRFT ISRDNSKNTL YLQMNSLRAE DTAVYYCAKP HYDGYYHFFD SWGQGTLVTV SSVEPKSADK THTCPPCPAP EAAGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLSCAVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLVSKLTV DKSRWQQGNV FSCSVMHEAL HNRYTQKSLS LSPGKTRIDENT-A amino acid sequence of the third polypeptide chain (SEQ ID NO: 107) as follows: DIQMTQSPSS LSASVGDRVT ITCKGSQDIN NYLAWYQQKP GKAPKLLIYN TDILHTGVPS RFSGSGSGTD FTFTISSLQP EDIATYYCYQ YNNGYTFGCG TKVEIKGGGG SGGGGSGGGG SQVQLVESGG GVVQPGRSLR LSCAASGFTF SDFGMNWVRQ APGKCLEWVA LIYYDGSNKF YADSVKGRFT ISRDNSKNTL YLQMNSLRAE DTAVYYCAKP HYDGYYHFFD SWGQGTLVTV SSVEPKSADK THTCPPCPAP EAAGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLSCAVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLVSKLTV DKSRWQQGNV FSCSVMHEAL HNRYTQKSLS LSPGK

TRIDENT-BTRIDENT-B

TRIDENT-B的第一多肽鏈的氨基酸序列與TRIDENT-A的第一多肽鏈的氨基酸序列相同(SEQ ID NO:105 )。TRIDENT-B的第二多肽鏈的氨基酸序列與TRIDENT-A的第二多肽鏈的氨基酸序列相同(SEQ ID NO:106 )。The amino acid sequence of the first polypeptide chain of TRIDENT-B is identical to the amino acid sequence of the first polypeptide chain of TRIDENT-A ( SEQ ID NO: 105 ). The amino acid sequence of the second polypeptide chain of TRIDENT-B is identical to the amino acid sequence of the second polypeptide chain of TRIDENT-A ( SEQ ID NO: 106 ).

TRIDENT-B的第三多肽鏈的氨基酸序列(SEQ ID NO:108 )如下所示： QVQLVESGGG VVQPGRSLRL SCAASGFTFS DFGMNWVRQA PGKGLEWVAL IYYDGSNKFY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKPH YDGYYHFFDS WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP SNTKVDKRVE PKSCDKTHTC PPCPAPEAAG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRE EMTKNQVSLS CAVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL VSKLTVDKSR WQQGNVFSCS VMHEALHNRY TQKSLSLSPG KTRIDENT-B amino acid sequence of the third polypeptide chain (SEQ ID NO: 108) as follows: QVQLVESGGG VVQPGRSLRL SCAASGFTFS DFGMNWVRQA PGKGLEWVAL IYYDGSNKFY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKPH YDGYYHFFDS WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP SNTKVDKRVE PKSCDKTHTC PPCPAPEAAG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRE EMTKNQVSLS CAVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL VSKLTVDKSR WQQGNVFSCS VMHEALHNRY TQKSLSLSPG K

TRIDENT-B的第四多肽鏈的氨基酸序列(SEQ ID NO:109 )如下所示： DIQMTQSPSS LSASVGDRVT ITCKGSQDIN NYLAWYQQKP GKAPKLLIYN TDILHTGVPS RFSGSGSGTD FTFTISSLQP EDIATYYCYQ YNNGYTFGQG TKVEIKRTVA APSVFIFPPS DEQLKSGTAS VVCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL SKADYEKHKV YACEVTHQGL SSPVTKSFNR GECTRIDENT-B amino acid sequence of a fourth polypeptide chain (SEQ ID NO: 109) as follows: DIQMTQSPSS LSASVGDRVT ITCKGSQDIN NYLAWYQQKP GKAPKLLIYN TDILHTGVPS RFSGSGSGTD FTFTISSLQP EDIATYYCYQ YNNGYTFGQG TKVEIKRTVA APSVFIFPPS DEQLKSGTAS VVCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL SKADYEKHKV YACEVTHQGL SSPVTKSFNR GEC

TRIDENT-CTRIDENT-C

TRIDENT-C的第一多肽鏈的氨基酸序列(SEQ ID NO:110 )如下所示(抗-ROR1-VL(14)以底線表示)： QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFSGSS SGADWYLTIS SLQSEDEADY YCGTDYPGNY LFGGGTQLTV LG GGGSGGGG EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMNWVRQA PGKGLEWVGR IRSKYNNYAT YYADSVKGRF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVS WFAYWGQGTL VTVSSASTKG EVAACEKEVA ALEKEVAALE KEVAALEKGG GDKTHTCPPC PAPEAAGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLWCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGKThe amino acid sequence of the first polypeptide chain of TRIDENT-C ( SEQ ID NO: 110 ) is shown below (anti-ROR1-VL (14) is indicated by the bottom line): QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFSGSS SGADWYLTIS SLQSEDEADY YCGTDYPGNY LFGGGTQLTV LG GGGSGGGG EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMNWVRQA PGKGLEWVGR IRSKYNNYAT YYADSVKGRF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVS WFAYWGQGTL VTVSSASTKG EVAACEKEVA ALEKEVAALE KEVAALEKGG GDKTHTCPPC PAPEAAGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLWCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK

TRIDENT-C的第二多肽鏈的氨基酸序列(SEQ ID NO:111 )如下所示(抗-ROR1-VH(8)以底線表示)： QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGG QE QLVESGGGLV QPGGSLRLSC AASGFTFSDY YMSWIRQAPG KGLEWVATIY PSSGKTYYAD SAKGRLTISS DNAKDSLYLQ MNSLRAEDTA VYYCTRDSYA DDAALFDIWG QGTTVTVSS A STKGKVAACK EKVAALKEKV AALKEKVAAL KEThe amino acid sequence of the second polypeptide chain of TRIDENT-C ( SEQ ID NO: 111 ) is shown below (anti-ROR1-VH(8) is indicated by the bottom line): QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGG QE QLVESGGGLV QPGGSLRLSC AASGFTFSDY YMSWIRQAPG KGLEWVATIY PSSGKTYYAD SAKGRLTISS DNAKDSLYLQ MNSLRAEDTA VYYCTRDSYA DDAALFDIWG QGTTVTVSS A STKGKVAACK EKVAALKEKV AALKEKVAAL KE

TRIDENT-C的第三多肽鏈的氨基酸序列與上述提供的TRIDENT-A的第三多肽鏈的氨基酸序列相同(SEQ ID NO:107 )。The amino acid sequence of the third polypeptide chain of TRIDENT-C is identical to the amino acid sequence of the third polypeptide chain of TRIDENT-A provided above ( SEQ ID NO: 107 ).

TRIDENT-DTRIDENT-D

TRIDENT-D的第一和第二多肽鏈與TRIDENT-C的第一和第二多肽鏈一致。因此，TRIDENT-D的第一多肽鏈的氨基酸序列是上述提供的SEQ ID NO:110 ，並且TRIDENT-D的第二多肽鏈的氨基酸序列是上述提供的SEQ ID NO:111 。TRIDENT-D的第三和第四多肽鏈與上述提供的TRIDENT-B的第三和第四多肽鏈一致(分別為SEQ ID NO:104 和SEQ ID NO:105 )。The first and second polypeptide chains of TRIDENT-D are identical to the first and second polypeptide chains of TRIDENT-C. Thus, the amino acid sequence of the first polypeptide chain of TRIDENT-D is SEQ ID NO: 110 provided above, and the amino acid sequence of the second polypeptide chain of TRIDENT-D is SEQ ID NO: 111 provided above. The third and fourth polypeptide chains of TRIDENT-D are identical to the third and fourth polypeptide chains of TRIDENT-B provided above ( SEQ ID NO: 104 and SEQ ID NO: 105, respectively ).

鑒於本文提供的教導，應當理解，可以利用不同的結構域取向、VH結構域、VL結構域、連接體、和/或異源二聚體促進結構域來生成可選的ROR1 x CD3 x CD8三價結合分子。例如，使用不同的抗-ROR1-VL和/或VH結構域生成TRIDENT-A和TRIDENT-C(見例如表 10 )。此外，本文提供的優化的抗-ROR1-VL和/或VH結構域中的任一個可以用於替代抗-ROR1-VL(14)和抗-ROR1-VH(8)，以生成供選擇的分子。In view of the teachings provided herein, it will be appreciated that different domain orientations, VH domains, VL domains, linkers, and/or heterodimer promoting domains can be utilized to generate alternative ROR1 x CD3 x CD8 III The valence binds to the molecule. For example, different anti-ROR1-VL and/or VH domains are used to generate TRIDENT-A and TRIDENT-C (see, eg, Table 10 ). Furthermore, any of the optimized anti-ROR1-VL and/or VH domains provided herein can be used in place of anti-ROR1-VL (14) and anti-ROR1-VH (8) to generate alternative molecules. .

XX .. 製備方法Preparation

如本領域所熟知的，本發明的ROR1-結合分子最優選通過編碼這類多肽的核酸分子的重組表達而產生。As is well known in the art, the ROR1-binding molecules of the invention are most preferably produced by recombinant expression of a nucleic acid molecule encoding such a polypeptide.

本發明的多肽可以使用肽固相合成方法被方便地製備(Merrifield, B. (1986) “Solid Phase Synthesis ,” Science 232(4748):341-347；Houghten, R.A. (1985) “General Method For The Rapid Solid-Phase Synthesis Of Large Numbers Of Peptides: Specificity Of Antigen-Antibody Interaction At The Level Of Individual Amino Acids ,” Proc. Natl. Acad. Sci. (U.S.A.) 82(15):5131-5135；Ganesan, A. (2006) “Solid-Phase Synthesis In The Twenty-First Century ,” Mini Rev. Med. Chem. 6(1):3-10)。The polypeptide of the present invention can be conveniently prepared using a peptide solid phase synthesis method (Merrifield, B. (1986) "S olid Phase Synthesis ," Science 232 (4748): 341-347; Houghten, RA (1985) " General Method For The Rapid Solid-Phase Synthesis Of Large Numbers Of Peptides: Specificity Of Antigen-Antibody Interaction At The Level Of Individual Amino Acids ," Proc. Natl. Acad. Sci. (USA) 82(15): 5131-5135; Ganesan, A (2006) " Solid-Phase Synthesis In The Twenty-First Century ," Mini Rev. Med. Chem. 6(1): 3-10).

在可選的方案中，可重組製備抗體和使用本領域已知的任何方法表達。可如下經重組製備抗體：首先從宿主動物分離製備的抗體，獲得基因序列，和使用基因序列在宿主細胞(例如，CHO細胞)中重組表達抗體。可採用的另一方法是在植物(例如，煙草)或轉基因奶中表達抗體序列。用於在植物或奶中重組表達抗體的適當的方法已經被公開了(見，例如Peeters等(2001) “Production Of Antibodies And Antibody Fragments In Plants ,” Vaccine 19:2756；Lonberg, N.等(1995) “Human Antibodies From Transgenic Mice ,” Int. Rev. Immunol 13:65-93；和Pollock等(1999) “Transgenic Milk As A Method For The Production Of Recombinant Antibodies ,” J. Immunol Methods 231:147-157)。製備抗體衍生物，例如，人源化抗體、單鏈抗體等的適當的方法是本領域已知的，並且已經在上面描述過了。在另一可選方案中，可通過噬菌體展示技術重組製備抗體(見，例如，美國專利號5,565,332、5,580,717、5,733,743、6,265,150；和Winter, G.等(1994) “Making Antibodies By Phage Display Technology ,” Annu. Rev. Immunol. 12.433-455)。In an alternative, the antibody can be recombinantly produced and expressed using any method known in the art. The antibody can be prepared recombinantly by first isolating the prepared antibody from the host animal, obtaining the gene sequence, and recombinantly expressing the antibody in a host cell (e.g., CHO cell) using the gene sequence. Another method that can be employed is to express antibody sequences in plants (eg, tobacco) or transgenic milk. Suitable methods for recombinant expression of antibodies in plants or milk have been disclosed (see, for example, Peeters et al. (2001) " Production Of Antibodies And Antibody Fragments In Plants ," Vaccine 19: 2756; Lonberg, N. et al. (1995). " Human Antibodies From Transgenic Mice ," Int. Rev. Immunol 13:65-93; and Pollock et al. (1999) " Transgenic Milk As A Method For The Production Of Recombinant Antibodies ," J. Immunol Methods 231: 147-157) . Suitable methods for preparing antibody derivatives, e.g., humanized antibodies, single chain antibodies, and the like, are known in the art and have been described above. In another alternative, the antibody can be recombinantly produced by phage display technology (see, e.g., U.S. Patent Nos. 5,565,332, 5,580,717, 5,733,743, 6,265,150; and Winter, G. et al. (1994) "making Antibodies By Phage Display Technology ," Annu. Rev. Immunol. 12.433-455).

可以通過許多合適的方法中的任何一種將含有感興趣的多核苷酸(例如，編碼本發明的ROR1-結合分子的多肽鏈的多核苷酸)的載體引入到宿主細胞中，所述適當手段包括電穿孔；採用氯化鈣、銣氯化物、磷酸鈣、DEAE-葡聚糖或其他物質的轉染；微彈轟擊(microprojectile bombardment)；脂轉染；和感染(例如，在載體是感染劑諸如痘苗病毒的情況下)。引入載體或多核苷酸的選擇常常取決於宿主細胞的特徵。A vector comprising a polynucleotide of interest (eg, a polynucleotide encoding a polypeptide chain of a ROR1-binding molecule of the invention) can be introduced into a host cell by any of a number of suitable methods, including: Electroporation; transfection with calcium chloride, cesium chloride, calcium phosphate, DEAE-dextran or other substances; microprojectile bombardment; lipofection; and infection (eg, in vehicles such as infectious agents) In the case of vaccinia virus). The choice of introducing a vector or polynucleotide will often depend on the characteristics of the host cell.

能夠過表達異源DNA的任何宿主細胞均可用於表達感興趣的多肽或蛋白質的目的。合適的哺乳動物宿主細胞的非限制性例子包括但不限於COS、HeLa和CHO細胞。Any host cell capable of overexpressing heterologous DNA can be used for the purpose of expressing a polypeptide or protein of interest. Non-limiting examples of suitable mammalian host cells include, but are not limited to, COS, HeLa, and CHO cells.

本發明包括包含本發明的ROR1-結合分子的氨基酸序列的多肽。可通過本領域已知的程式製備本發明的多肽。可通過抗體的蛋白水解或其他降解、如上述的重組方法(即，單個或融合多肽)或化學合成來產生多肽。抗體的多肽，尤其地，上至約50個氨基酸的較短的多肽，通過化學合成被常規製備。化學合成方法在本領域中是已知的，並且是商業上可得到的。The invention includes polypeptides comprising the amino acid sequence of a ROR1-binding molecule of the invention. The polypeptides of the invention can be prepared by procedures known in the art. The polypeptide can be produced by proteolysis or other degradation of the antibody, such as the recombinant methods described above (ie, single or fusion polypeptides) or by chemical synthesis. Polypeptides of antibodies, in particular, shorter polypeptides up to about 50 amino acids, are routinely prepared by chemical synthesis. Chemical synthesis methods are known in the art and are commercially available.

本發明包括ROR1-結合分子的變體，包括不明顯影響這類分子的特性的功能上等同的多肽以及具有增強的或降低的活性的變體。多肽的修飾在本領域中是常規操作，因而不需要在本文詳細描述。修飾的多肽的實例包括這樣的多肽：其具有氨基酸殘基的保守取代、未明顯有害改變功能活性的氨基酸的一個或多個缺失或添加，或使用化學類似物。可彼此保守取代的氨基酸殘基包括但不限於：甘氨酸/丙氨酸；絲氨酸/蘇氨酸；纈氨酸/異亮氨酸/亮氨酸；天冬醯胺/穀氨醯胺；天冬氨酸/谷氨酸；賴氨酸/精氨酸；和苯基丙氨酸/酪氨酸。這些多肽還包括糖基化和非糖基化多肽以及具有其它翻譯後修飾的多肽，所述其它翻譯後修飾，諸如例如，用不同糖進行糖基化、乙醯化和磷酸化。優選地，氨基酸取代應該是保守的，即，取代的氨基酸會具有與原始氨基酸類似的化學性質。這樣的保守取代在本領域中是已知的，並且已經在上文提供實例。氨基酸修飾範圍可從改變或修飾一個或多個氨基酸到區域諸如可變結構域的完全重新設計(redesign)。可變結構域中的變化可改變結合親和力和/或特異性。修飾的其它方法包括利用本領域中已知的偶合技術，包括但不限於酶促手段、氧化取代和螯合。修飾可用於例如，連接標籤用於免疫分析，諸如連接放射性部分用於放射免疫分析。修飾的多肽利用本領域中確定的方法製備，並且可利用本領域中已知的標準測定來篩選。The invention includes variants of ROR1-binding molecules, including functionally equivalent polypeptides that do not significantly affect the properties of such molecules, as well as variants with enhanced or reduced activity. Modification of the polypeptide is routine in the art and thus need not be described in detail herein. Examples of modified polypeptides include polypeptides that have conservative substitutions of amino acid residues, one or more deletions or additions of amino acids that are not significantly detrimental to functional activity, or the use of chemical analogs. Amino acid residues which may be conservatively substituted with each other include, but are not limited to, glycine/alanine; serine/threonine; valine/isoleucine/leucine; aspartame/glutamine; Lysine/glutamic acid; lysine/arginine; and phenylalanine/tyrosine. These polypeptides also include glycosylated and non-glycosylated polypeptides as well as polypeptides having other post-translational modifications, such as, for example, glycosylation, acetylation, and phosphorylation with different sugars. Preferably, the amino acid substitutions should be conservative, i.e., the substituted amino acids will have similar chemical properties as the original amino acids. Such conservative substitutions are known in the art and examples have been provided above. Amino acid modifications can range from alteration or modification of one or more amino acids to complete redesign of regions such as variable domains. Changes in the variable domain can alter binding affinity and/or specificity. Other methods of modification include the use of coupling techniques known in the art including, but not limited to, enzymatic means, oxidative substitution, and chelation. Modifications can be used, for example, to link tags for immunoassays, such as ligation of radioactive portions for radioimmunoassay. Modified polypeptides are prepared using methods established in the art and can be screened using standard assays known in the art.

本發明包括融合蛋白，所述融合蛋白包括本發明的優化的抗-ROR1-VL和/或VH中的一種或多種。在一個實施方式中，提供了融合多肽，其包括輕鏈、重鏈或輕鏈和重鏈二者。在另一實施方式中，提供的融合多肽包括輕鏈、重鏈或輕鏈和重鏈二者。在另一實施方式中，融合多肽包含異源免疫球蛋白恒定區。在另一實施方式中，融合多肽包含由公共保藏的雜交瘤產生的抗體的輕鏈可變結構域和重鏈可變結構域。為了本發明的目的，抗體融合蛋白包含特異性結合ROR1的一個或多個多肽結構域和其在天然分子中未連接的另外的氨基酸序列，所述另外的氨基酸序列例如，異源序列或來自另一區域的同源序列。The invention includes a fusion protein comprising one or more of the optimized anti-ROR1-VL and/or VH of the invention. In one embodiment, a fusion polypeptide is provided that includes a light chain, a heavy chain, or both a light chain and a heavy chain. In another embodiment, a fusion polypeptide is provided that includes a light chain, a heavy chain, or both a light chain and a heavy chain. In another embodiment, the fusion polypeptide comprises a heterologous immunoglobulin constant region. In another embodiment, the fusion polypeptide comprises a light chain variable domain and a heavy chain variable domain of an antibody produced by a publicly deposited hybridoma. For the purposes of the present invention, an antibody fusion protein comprises one or more polypeptide domains that specifically bind to ROR1 and additional amino acid sequences that are not linked in a native molecule, eg, a heterologous sequence or from another A homologous sequence of a region.

本發明尤其包括與診斷或治療部分綴合的ROR1-結合分子(例如，抗體、雙抗體、三價結合分子等)。出於診斷目的，本發明的ROR1-結合分子可以與可檢測物質偶聯(couple)。這類ROR1-結合分子可用於監測和/或預測疾病的發展或進展，作為臨床檢測程式的一部分，例如確定特定治療的效力。可檢測物質的實例包括各種酶(例如辣根過氧化物酶、β-半乳糖苷酶等)、輔基(例如鏈酶抗生物素/生物素)、螢光物質(例如7-羥基香豆素、螢光素或藻紅素)、發光物質(例如發光氨)、生物發光物質(例如螢光素酶或水母發光蛋白)、放射性物質(例如碳-14、錳-54、鍶-85或鋅-65)、正電子發射金屬和非放射性順磁金屬離子。可檢測物質可以使用本領域已知的技術與ROR1-結合分子直接偶聯或綴合，或通過中間體(例如，連接體)與ROR1-結合分子間接偶聯或綴合。The invention includes, inter alia, ROR1-binding molecules (eg, antibodies, diabodies, trivalent binding molecules, etc.) conjugated to a diagnostic or therapeutic moiety. For diagnostic purposes, the ROR1-binding molecules of the invention can be coupled to a detectable substance. Such ROR1-binding molecules can be used to monitor and/or predict the progression or progression of a disease as part of a clinical testing program, such as determining the efficacy of a particular treatment. Examples of detectable substances include various enzymes (for example, horseradish peroxidase, β-galactosidase, etc.), prosthetic groups (such as streptavidin/biotin), and fluorescent substances (for example, 7-hydroxycoumarin) , luciferin or phycoerythrin), luminescent substances (such as luminescent ammonia), bioluminescent substances (such as luciferase or aequorin), radioactive substances (such as carbon-14, manganese-54, strontium-85 or Zinc-65), positron-emitting metals and non-radioactive paramagnetic metal ions. The detectable substance can be directly coupled or conjugated to the ROR1-binding molecule using techniques known in the art, or indirectly coupled or conjugated to the ROR1-binding molecule via an intermediate (eg, a linker).

出於治療目的，本發明的ROR1-結合分子可以與治療部分如細胞毒素(例如細胞生長抑制劑或殺細胞劑)、治療劑或放射性金屬離子(例如α-發射體)綴合。細胞毒素或細胞毒性劑包括對細胞有害的任何試劑，例如假單胞菌外毒素、白喉毒素、肉毒桿菌毒素A至F、蓖麻毒蛋白相思豆毒蛋白、肥皂草毒蛋白和這些試劑的細胞毒性片段。治療劑包括具有預防性或治療性治療病症的治療效果的任何試劑。這樣的治療劑可以是化學治療劑、蛋白質或多肽治療劑，並且包括具有所需生物活性和/或改變給定生物應答的治療劑。治療劑的實例包括烷化劑、血管生成抑制劑、抗有絲***劑、激素治療劑和用於治療細胞增殖性病症的抗體。治療部分可以使用本領域已知的技術與ROR1-結合分子直接偶聯或綴合，或通過中間體(例如，連接體)與ROR1-結合分子或間接偶聯或綴合。For therapeutic purposes, the ROR1-binding molecules of the invention can be conjugated to a therapeutic moiety, such as a cytotoxin (eg, a cytostatic or cytocidal), a therapeutic agent, or a radioactive metal ion (eg, an alpha-emitter). Cytotoxins or cytotoxic agents include any agent that is harmful to cells, such as Pseudomonas exotoxin, diphtheria toxin, botulinum toxin A to F, ricin acacia, saponin, and these agents. Cytotoxic fragment. Therapeutic agents include any agent that has a therapeutic effect in the prophylactic or therapeutic treatment of a condition. Such therapeutic agents can be chemotherapeutic agents, protein or polypeptide therapeutics, and include therapeutic agents that have the desired biological activity and/or alter a given biological response. Examples of therapeutic agents include alkylating agents, angiogenesis inhibitors, anti-mitotic agents, hormonal therapeutics, and antibodies for treating cell proliferative disorders. The therapeutic moiety can be directly coupled or conjugated to the ROR1-binding molecule using techniques known in the art, or indirectly coupled or conjugated to the ROR1-binding molecule via an intermediate (eg, a linker).

本發明的The invention ROR1-ROR1- 結合分子的用途Use of binding molecules

本發明包括組合物，所述組合物包括藥物組合物，所述藥物組合物包含本發明的ROR1-結合分子(例如抗體、雙特異性抗體、雙特異性雙抗體、三價結合分子等)、來源於這些分子的多肽、包括編碼此類分子或多肽的序列的多核苷酸和本文所述的其它試劑。The invention includes a composition comprising a pharmaceutical composition comprising a ROR1-binding molecule of the invention (eg, an antibody, a bispecific antibody, a bispecific diabodies, a trivalent binding molecule, etc.), Polypeptides derived from such molecules, polynucleotides comprising sequences encoding such molecules or polypeptides, and other agents described herein.

如本文所提供的，包括本文提供的優化的抗-ROR1-VL和/或VH結構域的本發明的ROR1-結合分子具有結合存在於細胞表面上的ROR1並誘導抗體-依賴性細胞介導的細胞毒性(ADCC)和/或補體依賴性細胞毒性(CDC)和/或介導重定向細胞殺傷(例如重定向T-細胞細胞毒性)的能力。As provided herein, an ROR1-binding molecule of the invention comprising an optimized anti-ROR1-VL and/or VH domain provided herein has a binding to ROR1 present on the cell surface and induces antibody-dependent cell mediated Cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) and/or the ability to mediate redirected cell killing (eg, redirecting T-cell cytotoxicity).

因此，包括本文提供的優化的抗-ROR1-VL和/或VH結構域的本發明的ROR1-結合分子具有治療與ROR1的表達相關或特徵在於ROR1的表達的任何疾病或病況的能力。如上所述，ROR1是在許多血液和實體惡性腫瘤中表達的癌-胚抗原，其與表現出較低分化形態的高級腫瘤相關，並且與差的臨床結果相關(見例如， Zhang, S.,等(2012) “The Onco-Embryonic Antigen ROR1 Is Expressed by a Variety of Human Cancers, ” Am J. Pathol. 6:1903-1910；Zhang, H.等(2014) “ROR1 Expression Correlated With Poor Clinical Outcome In Human Ovarian Cancer ,” Sci Rep. 4:5811)。因此，沒有限制地，本發明的ROR1-結合分子可用於診斷或治療癌症，尤其是以ROR1的表達為特徵的癌症。Thus, a ROR1-binding molecule of the invention comprising an optimized anti-ROR1-VL and/or VH domain provided herein has the ability to treat any disease or condition associated with or characterized by expression of ROR1. As noted above, ROR1 is a cancer-embryo antigen expressed in many blood and solid malignancies that is associated with advanced tumors that exhibit a lower differentiated morphology and is associated with poor clinical outcomes (see, for example, Zhang, S., Etc. (2012) “ The Onco-Embryonic Antigen ROR1 Is Expressed by a Variety of Human Cancers, ” Am J. Pathol. 6:1903-1910; Zhang, H. et al. (2014) “ ROR1 Expression Correlated With Poor Clinical Outcome In Human Ovarian Cancer ,” Sci Rep. 4:5811). Thus, without limitation, the ROR1-binding molecules of the invention can be used to diagnose or treat cancer, particularly cancers characterized by expression of ROR1.

可以通過本發明的ROR1-結合分子治療的癌症包括以存在癌細胞為特徵的癌症，所述癌細胞選自由以下的細胞組成的組：腎上腺腫瘤、AIDS相關的癌症、軟組織腺泡狀肉瘤、星形細胞瘤、腎上腺癌、膀胱癌、骨癌、腦和脊髓癌、轉移性腦腫瘤、B細胞癌、乳腺癌、頸動脈體瘤、宮頸癌、軟骨肉瘤、脊索瘤、嫌色細胞腎細胞癌、透明細胞癌、結腸癌、結直腸癌、皮膚良性纖維組織細胞瘤、促結締組織增生小圓細胞瘤、室管膜細胞瘤、尤文氏瘤、骨外黏液樣軟骨肉瘤、不完全性骨纖維生成、骨的纖維發育異常、膽囊癌或膽管癌、胃癌、妊娠滋養層疾病、生殖細胞瘤、頭頸癌、血液學惡性腫瘤、肝細胞癌、胰島細胞腫瘤、卡波西氏肉瘤、腎癌、白血病、脂肪肉瘤/惡性的脂肪瘤、肝癌、淋巴瘤、肺癌、成神經管細胞瘤、黑素瘤、腦膜瘤、多發性內分泌瘤、多發性骨髓瘤、骨髓增生異常綜合征、成神經細胞瘤、神經內分泌腫瘤、卵巢癌、胰腺癌、甲狀腺乳頭狀癌、甲狀旁腺腫瘤、兒科癌症、周圍神經鞘瘤、嗜鉻細胞瘤、垂體瘤、***癌、眼色素層後黑素瘤、腎轉移性癌症、橫紋肌樣瘤、橫紋肌肉瘤、肉瘤、皮膚癌、軟組織肉瘤、鱗狀細胞癌、胃癌、滑膜肉瘤、睾丸癌、胸腺癌、胸腺瘤、甲狀腺轉移性癌和子宮癌。Cancers which can be treated by the ROR1-binding molecules of the present invention include cancers characterized by the presence of cancer cells selected from the group consisting of adrenal tumors, AIDS-related cancers, soft tissue acinar sarcomas, stars Agonoma, adrenal, bladder, bone, brain and spinal cord cancer, metastatic brain tumor, B cell carcinoma, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, chromophobe renal cell carcinoma , clear cell carcinoma, colon cancer, colorectal cancer, benign fibrous histiocytoma of the skin, connective tissue hyperplasia, small round cell tumor, ependymoma, Ewing's tumor, extra-muscular mucinous sarcoma, incomplete bone fiber Formation, bone dysplasia, gallbladder or cholangiocarcinoma, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hematological malignancy, hepatocellular carcinoma, islet cell tumor, Kaposi's sarcoma, kidney cancer, Leukemia, liposarcoma/malignant lipoma, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasms, multiple Myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid carcinoma, parathyroid tumor, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor , prostate cancer, melanoma of the pigmented layer, renal metastatic cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, synovial sarcoma, testicular cancer, thymus, thymus Tumor, metastatic thyroid cancer and uterine cancer.

尤其地，本發明的ROR1-結合分子可用於治療腎上腺癌、膀胱癌、乳腺癌、結直腸癌、胃癌、惡性膠質瘤、腎癌、非小細胞肺癌、急性淋巴細胞白血病、急性骨髓性白血病、慢性淋巴細胞白血病、慢性骨髓性白血病、毛細胞白血病、伯基特淋巴瘤、彌散性大B細胞淋巴瘤、濾泡淋巴瘤、套細胞淋巴瘤、邊緣區淋巴瘤、非霍奇金淋巴瘤、小淋巴細胞淋巴瘤、多發性骨髓瘤、黑素瘤、卵巢癌、胰腺癌、***癌、皮膚癌、腎細胞癌、睾丸癌和子宮癌。In particular, the ROR1-binding molecules of the invention can be used for the treatment of adrenal, bladder, breast, colorectal, gastric, glioblastoma, renal, non-small cell lung, acute lymphoblastic, acute myeloid leukemia, Chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal lymphoma, non-Hodgkin's lymphoma, Small lymphocytic lymphoma, multiple myeloma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, renal cell carcinoma, testicular cancer, and uterine cancer.

本發明的雙特異性ROR1-結合分子通過促進對表達此類分子(例如CD2、CD3、CD8、CD16、T細胞受體(TCR)、NKG2D等)的第二特異性的腫瘤細胞的重定向殺傷來增加由ROR1提供的癌症治療。這樣的ROR1-結合分子尤其可用於癌症的治療。The bispecific ROR1-binding molecules of the invention are directed to promote retinal killing of tumor cells that express a second specificity for expression of such molecules (eg, CD2, CD3, CD8, CD16, T cell receptor (TCR), NKG2D, etc.) To increase the cancer treatment provided by ROR1. Such ROR1-binding molecules are especially useful for the treatment of cancer.

除了它們在治療中的實用性，本發明的ROR1-結合分子可以被可檢測地標記並用於癌症的診斷或用於腫瘤和腫瘤細胞的成像。In addition to their utility in therapy, the ROR1-binding molecules of the invention can be detectably labeled and used in the diagnosis of cancer or in the imaging of tumors and tumor cells.

XIIXII .. 藥物組合物Pharmaceutical composition

本發明的組合物包括原料藥物組合物，其可用於製造藥物組合物(例如，不純的或非無菌組合物)和可用於製備單位劑型的藥物組合物(即，適於施用至受試者或患者的組合物)。這類組合物包括預防上或治療上有效量的本發明的ROR1-結合分子，或這類劑和藥學上可接受的載體的組合。優選地，本發明的組合物包括預防上或治療上有效量的本發明的ROR1-結合分子和藥學上可接受的載體。本發明也包括這類藥物組合物，其另外包括對特定的癌症抗原特異性的第二治療性抗體(例如，腫瘤特異性單克隆抗體)，和藥學上可接受的載體。The compositions of the present invention comprise a pharmaceutical composition of the starting material which can be used in the manufacture of a pharmaceutical composition (for example, an impure or non-sterile composition) and a pharmaceutical composition which can be used in the preparation of a unit dosage form (ie, suitable for administration to a subject or Patient's composition). Such compositions include a prophylactically or therapeutically effective amount of a ROR1-binding molecule of the invention, or a combination of such agents and a pharmaceutically acceptable carrier. Preferably, the compositions of the invention comprise a prophylactically or therapeutically effective amount of a ROR1-binding molecule of the invention and a pharmaceutically acceptable carrier. The invention also includes such pharmaceutical compositions additionally comprising a second therapeutic antibody (e.g., a tumor-specific monoclonal antibody) specific for a particular cancer antigen, and a pharmaceutically acceptable carrier.

在具體的實施方案中，術語“藥學上可接受的”表示獲得聯邦政府或州政府管理機構的許可或列於美國藥典(U.S. Pharmacopeia)或其他通常獲得認可的藥典中，供用於動物，特別是用於人類。術語“載體”指與治療劑一起施用的稀釋劑、佐劑(例如弗氏佐劑(完全和不完全)、賦形劑或媒介。一般而言，本發明組合物的成分被單獨提供或以單位劑型混合在一起，例如作為標明活性劑的量的密封容器中的凍乾粉或無水濃縮物，所述密封容器如安瓿或小袋(sachette)。當通過輸注施用組合物時，其可以用含有無菌的藥學級水或鹽水的輸注瓶分配。如果通過注射施用所述組合物，則可以提供一安瓿注射用無菌水或鹽水，以便可以在施用前混合所述成分。In a specific embodiment, the term "pharmaceutically acceptable" means obtaining permission from a federal or state government regulatory agency or listed in the US Pharmacopeia or other commonly recognized pharmacopoeia for use in animals, particularly Used in humans. The term "carrier" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete), excipient or vehicle) to be administered with a therapeutic agent. In general, the ingredients of the compositions of the present invention are provided separately or in The unit dosage forms are mixed together, for example as a lyophilized powder or a water-free concentrate in a sealed container in an amount indicating the active agent, such as an ampoule or sachette. When the composition is applied by infusion, it may be contained A sterile pharmaceutical grade water or saline infusion bottle is dispensed. If the composition is administered by injection, an ampoule of sterile water or saline for injection may be provided so that the ingredients can be mixed prior to administration.

本發明也提供藥物包裝或試劑盒，其包括一個或多個容器，所述容器填充本發明的ROR1-結合分子，單獨地或與這類藥學上可接受的載體一起。另外，用於治療疾病的一種或多種其他預防劑或治療劑也可包括在藥物包裝或試劑盒中。本發明也提供了這樣的藥物包裝或試劑盒，其包括一個或多個容器，所述容器填充本發明藥物組合物的一種或多種成分。任選地與這類容器(一個或多個)關聯的可以是管理藥物或生物產品的製造、使用或銷售的政府機構規定的形式的佈告(notice)，所述佈告反映了管理機構許可用於人類施用的製造、使用或銷售。The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with the ROR1-binding molecules of the invention, either alone or with such pharmaceutically acceptable carriers. Additionally, one or more additional prophylactic or therapeutic agents for treating the disease can also be included in the pharmaceutical pack or kit. The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) may be a notice in the form prescribed by a government agency that governs the manufacture, use or sale of a drug or biological product, the notice reflecting the regulatory agency's permission to use Manufacture, use or sale of human administration.

本發明提供了可用於上述方法的試劑盒。試劑盒可以包括本發明的任何ROR1-結合分子。試劑盒可在一個或多個容器中進一步包括可用於治療癌症的一種或多種其他預防劑和/或治療劑。The present invention provides kits that can be used in the above methods. Kits can include any of the ROR1-binding molecules of the invention. The kit may further comprise one or more additional prophylactic and/or therapeutic agents useful in the treatment of cancer in one or more containers.

XIII.XIII. 給藥方法Method of administration

通過向受試者施用有效量的本發明的融合蛋白或綴合分子或包括本發明的融合蛋白或綴合分子的藥學組合物，可以提供本發明的組合物用來治療、預防和改善與疾病、病症或感染相關的一種或多種症狀。在優選的方面，這類組合物基本上是純的(即，基本上不含限制其效果或產生不期望的副作用的物質)。在具體實施方式中，受試者是動物，優選哺乳動物，如非靈長類(例如牛、馬、貓科動物、犬科動物、齧齒動物等)或靈長類(例如，猴子，如食蟹猴、人等)。在優選的實施方式中，受試者是人。The compositions of the present invention can be provided for the treatment, prevention, and amelioration of diseases by administering to the subject an effective amount of a fusion protein or conjugate molecule of the invention or a pharmaceutical composition comprising a fusion protein or conjugate molecule of the invention One or more symptoms associated with the condition or infection. In a preferred aspect, such compositions are substantially pure (i.e., substantially free of materials that limit their effectiveness or produce undesirable side effects). In a specific embodiment, the subject is an animal, preferably a mammal, such as a non-primate (eg, cow, horse, feline, canine, rodent, etc.) or primate (eg, monkey, such as a food) Crab monkeys, people, etc.). In a preferred embodiment, the subject is a human.

各種送遞系統是已知的，並且可以用於施用本發明的組合物，例如封裝於脂質體中、微粒、微膠囊、能表達抗體或融合蛋白的重組細胞、受體介導的內吞作用(見，例如，Wu等(1987) “Receptor-Mediated In Vitro Gene Transformation By A Soluble DNA Carrier System,” J. Biol. Chem. 262:4429-4432)、構建核酸作為逆轉錄病毒或其他載體的一部分等。Various delivery systems are known and can be used to administer the compositions of the invention, for example, encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing antibodies or fusion proteins, receptor-mediated endocytosis (See, for example, Wu et al. (1987) "Receptor-Mediated In Vitro Gene Transformation By A Soluble DNA Carrier System," J. Biol. Chem. 262: 4429-4432), constructing nucleic acids as part of a retrovirus or other vector Wait.

施用本發明的分子的方法包括但不限於腸胃外施用(例如皮內、肌肉、腹腔內、靜脈內以及皮下)、硬膜外以及粘膜(例如鼻內和口腔途徑)。在具體實施方式中，本發明的ROR1-結合分子經肌肉、靜脈內或皮下施用。組合物可以通過任何方便途徑施用，例如通過輸注或彈丸注射、通過上皮或黏膜皮膚被覆(lining) (例如口腔粘膜、直腸和腸粘膜等)吸收，並且可以與其他生物活性劑一起施用。給藥可以是全身的或局部的。另外，也可以應用肺給藥，例如通過使用吸入器或噴霧器，並且與霧化劑一起配製。見，例如，美國專利號6,019,968、5,985,320、5,985,309、5,934,272、5,874,064、5,855,913、5,290,540和4,880,078；和PCT公佈號WO 92/19244、WO 97/32572、WO 97/44013、WO 98/31346和WO 99/66903，其每一篇通過引用以其整體併入本文。Methods of administering the molecules of the invention include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural, and mucosal (e.g., intranasal and buccal routes). In a specific embodiment, the ROR1-binding molecules of the invention are administered intramuscularly, intravenously or subcutaneously. The compositions can be administered by any convenient route, for example by infusion or bolus injection, by epithelial or mucosal skin lining (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and can be administered with other bioactive agents. Administration can be systemic or topical. In addition, pulmonary administration can also be applied, for example by using an inhaler or a nebulizer, and formulated with an aerosol. See, for example, U.S. Patent Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/ 66,903, each of which is incorporated herein in its entirety by reference.

本發明也使得本發明的ROR1-結合分子的製劑包裝在密封容器中，比如指示分子的數量的安瓿或小袋中。在一個實施方式中，這樣的分子作為凍幹無菌粉或無水濃縮物提供於密封容器中，並且可以用例如水或鹽水重構至適當濃度，用於施用於受試者。優選地，本發明的ROR1-結合分子作為無菌凍幹粉末提供在密封容器中。The invention also allows the formulation of the ROR1-binding molecules of the invention to be packaged in a sealed container, such as an ampoule or sachet indicating the number of molecules. In one embodiment, such a molecule is provided as a lyophilized sterile powder or anhydrous concentrate in a sealed container and can be reconstituted to a suitable concentration with, for example, water or saline for administration to a subject. Preferably, the ROR1-binding molecules of the invention are provided as a sterile lyophilized powder in a sealed container.

本發明的ROR1-結合分子的凍幹製劑應在它們的初始容器中儲存在2°C和8°C之間，並且分子應該在重構之後12小時內，優選地6小時內、5小時內、3小時內或1小時內施用。在可選的實施方式中，這樣的分子以液體形式提供在指示分子、融合蛋白或綴合分子的量和濃度的密封容器中。優選地，這類ROR1-結合分子在以液體形式提供時，被供應在密封容器中。The lyophilized preparations of the ROR1-binding molecules of the invention should be stored between 2 ° C and 8 ° C in their original container, and the molecules should be within 12 hours, preferably within 6 hours, within 5 hours after reconstitution Apply within 3 hours or within 1 hour. In an alternative embodiment, such molecules are provided in liquid form in a sealed container of the amount and concentration of the indicator molecule, fusion protein or conjugate molecule. Preferably, such ROR1-binding molecules are supplied in a sealed container when provided in liquid form.

可通過標準臨床技術確定本發明的這類製劑有效治療、預防或改善與病症相關的一個或多個症狀的量。製劑中採用的精確劑量還將取決於施用的路徑和病況的嚴重性，並且應根據從業者的判斷和每個患者的情況決定。有效的劑量可從源自體外或動物模型測試系統的劑量回應曲線推斷。Such formulations of the invention can be determined by standard clinical techniques to effectively treat, prevent or ameliorate the amount of one or more symptoms associated with the condition. The precise dose employed in the formulation will also depend on the route of administration and the severity of the condition and should be determined in accordance with the judgment of the practitioner and the condition of each patient. Effective doses can be inferred from dose response curves derived from in vitro or animal model test systems.

如本文所使用的，藥物組合物的“有效量 ”是足夠實現有益或期望的結果的量，所述結果包括但不限於臨床結果，比如減少源自疾病的症狀、減少感染的症狀(例如，病毒量、發燒、疼痛、敗血症等)或癌症的症狀(例如，癌細胞的增殖、腫瘤存在、腫瘤轉移等)，從而提高遭受疾的患者的生命品質，降低治療疾病需要的其他藥物治療的劑量、比如經靶向和/或內化增強另一藥物的作用、延遲疾病的進展，和/或延長個體的生存。As used herein, an " effective amount " of a pharmaceutical composition is an amount sufficient to achieve a beneficial or desired result, including but not limited to clinical outcomes, such as reducing symptoms from a disease, reducing symptoms of infection (eg, Symptoms of viral disease, fever, pain, sepsis, etc. or cancer (eg, proliferation of cancer cells, tumors, tumor metastasis, etc.), thereby improving the quality of life of patients suffering from disease and reducing the dose of other medications needed to treat the disease For example, targeting and/or internalizing enhances the action of another drug, delays the progression of the disease, and/or prolongs the survival of the individual.

有效量可以在一次或多次給藥中被施用。為了本發明的目的，藥物、化合物或藥物組合物的有效量是足以直接或間接減少病毒存在的增殖(或效應)並減少和/或延遲病毒疾病發展的量。在一些實施方案中，藥物、化合物或藥物組合物的有效量可聯合或不聯合另一藥物、化合物或藥物組合物實現。因此，“有效量”可在施用一種或多種化療劑的背景下考慮，並且如聯合一種或多種其他劑，可實現或實現期望的結果，則單劑可視為以有效量施用。儘管個體的需要不同，但是測定每種組分的有效量的最佳範圍是本領域技術人員已知的。An effective amount can be administered in one or more administrations. For the purposes of the present invention, an effective amount of a drug, compound or pharmaceutical composition is an amount sufficient to directly or indirectly reduce the proliferation (or effect) of the presence of the virus and to reduce and/or delay the progression of the viral disease. In some embodiments, an effective amount of a drug, compound, or pharmaceutical composition can be achieved with or without another drug, compound, or pharmaceutical composition. Thus, an "effective amount" can be considered in the context of administering one or more chemotherapeutic agents, and if one or more other agents are combined to achieve or achieve the desired result, a single dose can be considered to be administered in an effective amount. Although the needs of the individual are different, the optimal range for determining the effective amount of each component is known to those skilled in the art.

對於本發明包括的ROR1-結合分子，施用於患者的劑量優選基於接受受試者的體重(kg)確定。對於本發明包括的ROR1-結合分子，施用於患者的劑量通常為受試者體重的約0.01μg/kg至約30 mg/kg或更多。For the ROR1-binding molecules encompassed by the invention, the dosage administered to the patient is preferably determined based on the body weight (kg) of the subject. For the ROR1-binding molecules encompassed by the invention, the dosage administered to the patient will generally be from about 0.01 μg/kg to about 30 mg/kg or more of the subject's body weight.

可通過修飾比如，例如脂質化而增強分子的吸收和組織滲透，來減少或改變本發明的ROR1-結合分子的給藥劑量和頻率。The dosage and frequency of administration of the ROR1-binding molecules of the invention can be reduced or altered by modifying, for example, lipidation to enhance absorption and tissue penetration of the molecule.

對於用作單劑療法，可計算向患者施用的本發明的ROR1-結合分子的劑量。可選地，分子可與其他治療組合物聯合使用，並且向患者施用的劑量小於當所述分子用作單劑療法時的劑量。For use as a single dose therapy, the dose of the ROR1-binding molecule of the invention administered to a patient can be calculated. Alternatively, the molecule can be used in combination with other therapeutic compositions, and the dose administered to the patient is less than the dose when the molecule is used as a single dose of therapy.

本發明的藥物組合物可被局部施用至需要治療的區域；這可通過例如，但不限於下述方式實現：局部注入、通過注射、或通過植入物的手段，所述植入物是多孔的、非多孔的或膠狀材料，包括膜，比如矽橡膠膜或纖維。優選地，當施用本發明的分子時，必須注意使用不吸收該分子的材料。The pharmaceutical composition of the present invention can be topically applied to the area in need of treatment; this can be achieved, for example, but not limited to, by local injection, by injection, or by means of an implant, which is porous , non-porous or gel-like materials, including films such as silicone rubber films or fibers. Preferably, when administering the molecules of the invention, care must be taken to use materials that do not absorb the molecules.

本發明的組合物可以在泡狀體(vesicle)，尤其是脂質體中遞送(見Langer (1990) “New Methods Of Drug Delivery,”Science 249:1527-1533)；Treat等,在Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein和Fidler (編輯)，Liss, New York，pp. 353- 365 (1989)中；Lopez-Berestein, 同上, pp. 3 17-327).The compositions of the invention may be delivered in vesicles, especially liposomes (see Langer (1990) "New Methods Of Drug Delivery," Science 249: 1527-1533); Treat et al, in Liposomes in the Therapy Of Infectious Disease and Cancer, Lopez-Berestein and Fidler (ed.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 3 17-327).

當本發明的組合物是編碼本發明的ROR1-結合分子的核酸的情況下，核酸可被體內施用，以通過如下方式促進其編碼的ROR1-結合分子的表達：將其構建為適當的核酸表達載體的一部分並且施用其從而其成為細胞內的，例如，通過使用逆轉錄病毒載體(見美國專利號4,980,286)，或通過直接注射，或通過使用微粒轟擊(例如，基因槍；生物彈道技術(Biolistic), Dupont)，或用脂質或細胞表面受體或轉染試劑塗覆，或通過與已知進入核的同源框樣肽聯合施用(見例如，Joliot等(1991) “Antennapedia Homeobox Peptide Regulates Neural Morphogenesis,”Proc. Natl. Acad.Sci. (U.S.A.) 88:1864-1868)等。可選地，可以將核酸引入細胞內並通過同源重組整合到宿主細胞DNA中，以進行表達。Where the composition of the invention is a nucleic acid encoding a ROR1-binding molecule of the invention, the nucleic acid can be administered in vivo to facilitate expression of the encoded ROR1-binding molecule by constructing it into an appropriate nucleic acid expression. A portion of the vector and administered thereto such that it becomes intracellular, for example, by using a retroviral vector (see U.S. Patent No. 4,980,286), or by direct injection, or by using microprojectile bombardment (eg, gene gun; bio-ballistic technique (Biolistic) ), Dupont), or coated with lipid or cell surface receptors or transfection reagents, or by administration in combination with a homologous framework peptide known to enter the nucleus (see, for example, Joliot et al. (1991) “Antennapedia Homeobox Peptide Regulates Neural Morphogenesis, "Proc. Natl. Acad. Sci. (USA) 88: 1864-1868) and the like. Alternatively, the nucleic acid can be introduced into a cell and integrated into host cell DNA by homologous recombination for expression.

用治療或預防有效量的本發明的ROR1-結合分子對受試者的治療可包括單治療，或優選地，可包括一系列治療。在優選的實施例中，用這類雙抗體治療受試者每週一次，持續約1至10周，優選地2至8周，更優選地約3至7周，和甚至更優選地持續約4、5或6周。本發明的藥物組合物可以每天施用一次，其中這樣的給藥每週發生一次、每週發生兩次、每兩周發生一次、每月發生一次、每六周發生一次、每兩月發生一次、每年發生兩次或每年發生一次等。可選地，本發明的藥物組合物可每天施用兩次，其中這樣的給藥每週發生一次、每週發生兩次、每兩周發生一次、每月發生一次、每六周發生一次、每兩月發生一次、每年發生兩次或每年發生一次等。可選地，本發明的藥物組合物可每天施用三次，其中這樣的給藥每週發生一次、每週發生兩次、每兩周發生一次、每月發生一次、每六周發生一次，每兩月發生一次、每年發生兩次或每年發生一次等。還應當理解，用於治療的分子的有效劑量可以在特定治療過程中增加或減少。Treatment of a subject with a therapeutically or prophylactically effective amount of a ROR1-binding molecule of the invention can include a single treatment or, preferably, can include a series of treatments. In a preferred embodiment, the subject is treated once a week with such a diabody for about 1 to 10 weeks, preferably 2 to 8 weeks, more preferably about 3 to 7 weeks, and even more preferably for about 4, 5 or 6 weeks. The pharmaceutical composition of the present invention can be administered once a day, wherein such administration occurs once a week, twice a week, once every two weeks, once a month, once every six weeks, once every two months, It occurs twice a year or once a year. Alternatively, the pharmaceutical composition of the invention may be administered twice daily, wherein such administration occurs once a week, twice a week, once every two weeks, once a month, every six weeks, every time It occurs once every two months, twice a year, or once a year. Alternatively, the pharmaceutical composition of the present invention may be administered three times a day, wherein such administration occurs once a week, twice a week, once every two weeks, once a month, once every six weeks, every two times It occurs once a month, twice a year, or once a year. It will also be appreciated that the effective dosage of the molecule for treatment may be increased or decreased during a particular course of treatment.

實施例Example

現在已經大體上描述了本發明，通過參考下述實施例其將更容易理解。下述實施例闡釋了組合物在本發明的診斷或治療方法中的各種方法。實施例旨在闡釋但決不限制本發明的範圍。The invention has now been generally described, which will be more readily understood by reference to the following embodiments. The following examples illustrate various methods of the compositions in the methods of diagnosis or treatment of the present invention. The examples are intended to illustrate, but in no way limit, the scope of the invention.

實施例Example 11

抗anti- -ROR1-VL-ROR1-VL 和抗And anti -ROR1-VH-ROR1-VH 的優化Optimization

為了獲得對人ROR1表現出改善的親和力的優化的抗-ROR1抗體種類，對編碼親本抗-ROR1抗體VL和抗-ROR1-VH結構域的多核苷酸(即分別為抗-ROR1-VL或抗-ROR-VH)進行誘變。VL結構域變體被命名為“抗 -ROR1-VL(2) ”、“抗 -ROR1-VL(3) ”、“抗 -ROR1-VL(4) ”、“抗 -ROR1-VL(5) ”、“抗 -ROR1-VL(6) ”、“抗 -ROR1-VL(7) ”、“抗 -ROR1-VL(8) ”、“抗 -ROR1-VL(9) ”、“抗 -ROR1-VL(10) ”、“抗 -ROR1-VL(11) ”、“抗 -ROR1-VL(12) ”、“抗 -ROR1-VL(13) ”和“抗 -ROR1-VL(14) ”，並且VH結構域變體被命名為“抗 -ROR1-VH(1) ”、“抗 -ROR1-VH(2) ”、“抗 -ROR1-VH(3) ”、“抗 -ROR1-VH(4) ”、“抗 -ROR1-VH(5) ”、“抗 -ROR1-VH(6) ”和“抗 -ROR1-VH(7) ”。這些變體的氨基酸序列在上文被提供，突變和相應的SEQ ID NO 被總結在表 6 中。 To obtain an optimized anti-ROR1 antibody class that exhibits improved affinity for human ROR1, a polynucleotide encoding a parental anti-ROR1 antibody VL and an anti-ROR1-VH domain (ie, anti-ROR1-VL or Mutagenesis was carried out by anti-ROR-VH. VL domain variants were named " anti- ROR1-VL(2) ", " anti- ROR1-VL(3) ", " anti- ROR1-VL(4) ", " anti- ROR1-VL(5) ", anti- ROR1-VL(6) ", " anti- ROR1-VL(7) ", " anti- ROR1-VL(8) ", " anti- ROR1-VL(9) ", " anti- ROR1" -VL(10) ", " Anti- ROR1-VL(11) ", " Anti- ROR1-VL(12) ", " Anti- ROR1-VL(13) ", and " Anti- ROR1-VL(14) " And the VH domain variants are named " anti- ROR1-VH(1) ", " anti- ROR1-VH(2) ", " anti- ROR1-VH(3) ", " anti- ROR1-VH ( 4) ”, “ Anti- ROR1-VH(5) ”, “ Anti- ROR1-VH(6) ” and “ Anti- ROR1-VH(7) ”. The amino acid sequences of these variants are provided above, and the mutations and corresponding SEQ ID NOs are summarized in Table 6 .

產生了31種ROR1 × CD3雙特異性雙鏈共價結合的雙抗體，每種均具有對ROR1特異性的一個結合位點(其包括親本的和/或變異的抗-ROR1-VL和抗-ROR1-VH結構域)和對CD3具有特異性的一個結合位點(其包括CD3 mAb 1(D65G)的VL和VH結構域)。上文詳細提供了這些示例性ROR1 × CD3雙特異性雙鏈雙抗體的第一和第二多肽鏈的一般結構。在每種雙抗體(連續編號並命名為“DART-1 ”至“DART-31 ”)中存在的特定抗-ROR1-VL和抗-ROR1-VH結構域提供於表 7 中。這類雙抗體的CD3結合結構域是CD3 mAb 1的VL結構域(SEQ ID NO:75 )或抗-CD3 mAb 1(D65G)的VH結構域(SEQ ID NO:77 )。抗-ROR1結合結構域和抗-CD3結合結構域通過間插間隔體肽(連接體1)GGGSGGGG(SEQ ID NO:33 )彼此分開。 √: 相似的結合 ↑: 提高的結合 ↓: 降低的結合 -: 未確定Thirty-two ROR1 × CD3 bispecific double-stranded covalently bound diabodyes, each having a binding site specific for ROR1 (including parental and/or variant anti-ROR1-VL and anti-R) a -ROR1-VH domain) and a binding site specific for CD3 (which includes the VL and VH domains of CD3 mAb 1 (D65G)). The general structure of the first and second polypeptide chains of these exemplary ROR1 x CD3 bispecific double-chain diabodies is provided in detail above. The specific anti-ROR1-VL and anti-ROR1-VH domains present in each diabody (numbered consecutively and named " DART-1 " through " DART-31 ") are provided in Table 7 . The CD3 binding domain of such a diabody is the VL domain of CD3 mAb 1 ( SEQ ID NO: 75 ) or the VH domain of anti-CD3 mAb 1 (D65G) ( SEQ ID NO: 77 ). The anti-ROR1 binding domain and the anti-CD3 binding domain are separated from each other by an intervening spacer peptide (Linker 1) GGGSGGGG ( SEQ ID NO: 33 ). √: Similar combination ↑: Improved combination ↓: Reduced binding -: Not determined

DART-1DART-1

為了闡釋，DART-1包括親本抗-ROR1-VL和抗-ROR1-VL結構域。DART-1的氨基酸序列在下面提供。To illustrate, DART-1 includes the parental anti-ROR1-VL and anti-ROR1-VL domains. The amino acid sequence of DART-1 is provided below.

DART-1的第一多肽鏈的氨基酸序列(SEQ ID NO:112 )如下所示(親本抗-ROR1-VL以實底線表示；抗-CD3結合結構域以虛底線表示 )： QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFGSGS SSGADRYLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLG GGGSGGG GEVQLVESGG GLVQPGGSLR LSCAASGFTF STYAMNWVRQ APGKGLEWVG RIRSKYNNYA TYYADSVKGR FTISRDDSKN SLYLQMNSLK TEDTAVYYCV RHGNFGNSYV SWFAYWGQGT LVTVSS GGCG GGKVAALKEK VAALKEKVAA LKEKVAALKEThe amino acid sequence of the first polypeptide chain of DART-1 ( SEQ ID NO: 112 ) is shown below (parent anti-ROR1-VL is shown in solid bottom; anti-CD3 binding domain is indicated by dashed bottom line): QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFGSGS SSGADRYLTI SSLQSEDEAD YYCGTDYPGN YLFGGGTQLT VLG GGGSGGG G EVQLVESGG GLVQPGGSLR LSCAASGFTF STYAMNWVRQ APGKGLEWVG RIRSKYNNYA TYYADSVKGR FTISRDDSKN SLYLQMNSLK TEDTAVYYCV RHGNFGNSYV SWFAYWGQGT LVTVSS GGCG GGKVAALKEK VAALKEKVAA LKEKVAALKE

DART-1的第二多肽鏈的氨基酸序列(SEQ ID NO:113 )如下所示(親本抗-ROR1-VH以實底線表示；抗-CD3結合結構域以虛底線表示 )：QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGG QE QLVESGGGLV QPGGSLRLSC AASGFTFSDY YMSWVRQAPG KGLEWVATIY PSSGKTYYAD SVKGRFTISS DNAKNSLYLQ MNSLRAEDTA VYYCARDSYA DDAALFDIWG QGTTVTVSS G GCGGGEVAAL EKEVAALEKE VAALEKEVAA LEKThe amino acid sequence of the second polypeptide chain of DART-1 ( SEQ ID NO: 113 ) is shown below (parent anti-ROR1-VH is shown as a solid underline; anti-CD3 binding domain is indicated by a dashed bottom line): QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGG QE QLVESGGGLV QPGGSLRLSC AASGFTFSDY YMSWVRQAPG KGLEWVATIY PSSGKTYYAD SVKGRFTISS DNAKNSLYLQ MNSLRAEDTA VYYCARDSYA DDAALFDIWG QGTTVTVSS G GCGGGEVAAL EKEVAALEKE VAALEKEVAA LEK

包括變異的VL和VH結構域(即，抗-ROR1-VL(2)和抗-ROR1-VH(7))的代表性ROR1×CD3雙特異性雙鏈雙抗體DART-25的第一和第二多肽鏈的氨基酸序列在上文提供。First and first representative ROR1×CD3 bispecific double-stranded diabody DART-25 including variant VL and VH domains (ie, anti-ROR1-VL(2) and anti-ROR1-VH(7)) The amino acid sequence of the dipeptide chain is provided above.

通過ELISA檢驗DART-1至DART-31與可溶性人ROR1的結合。簡言之，用0.5μg/mL的His標記的可溶性人ROR1(“shROR1-His”，包含與His-Tag融合的人ROR1的胞外部分) 塗覆微量滴定板，洗滌板，並與所產生的雙抗體(DART-1至DART-31)之一的三倍連續稀釋物一起孵育。使用通過鏈酶抗生物素-HRP檢測的生物素化的抗-E/K螺旋第二抗體來評價與固定化ROR1結合的雙抗體的量。在板閱讀器上分析所有樣品，並生成結合曲線。相對於DART-1，DART-2至DART-31的結合總結於上面表 7 中。包括變異的VL和/或變異的VH結構域的一些雙抗體相對於DART-1顯示改善的結合，表明這類可變結構域是優化的。Binding of DART-1 to DART-31 to soluble human ROR1 was tested by ELISA. Briefly, 0.5 μg/mL His-labeled soluble human ROR1 ("shROR1-His", including the extracellular portion of human ROR1 fused to His-Tag) was coated with a microtiter plate, washed, and produced Incubate with a three-fold serial dilution of one of the diabody (DART-1 to DART-31). The amount of diabody bound to immobilized ROR1 was evaluated using a biotinylated anti-E/K helix secondary antibody detected by streptavidin-HRP. All samples were analyzed on a plate reader and a binding curve was generated. The binding of DART-2 to DART-31 relative to DART-1 is summarized in Table 7 above. Some diabodyes comprising a variant VL and/or a variant VH domain showed improved binding relative to DART-1, indicating that such variable domains are optimized.

使用Biacore分析研究DART-1至DART-6、DART-10至DART-17、DART-20至DART-33的結合動力學，其中使ROR1蛋白在固定化的雙抗體上通過。簡言之，每種雙抗體構建體被捕獲在固定化的抗-E/K-螺旋表面上，並與25和100nM的shROR1-His一起孵育，並且通過Biacore分析確定結合動力學。來自這些研究的計算的ka、kd和KD示於表7中。導致改善的結合的大多數突變位於CDR外部。尤其地，在這些研究中，存在於DART-2(抗-ROR1-VL(2))中的單R71W取代使結合增強超過兩倍。Binding kinetics of DART-1 to DART-6, DART-10 to DART-17, DART-20 to DART-33 were studied using Biacore analysis, wherein the ROR1 protein was passed over the immobilized diabody. Briefly, each diabody construct was captured on an immobilized anti-E/K-helix surface and incubated with 25 and 100 nM shROR1-His and binding kinetics were determined by Biacore analysis. The calculated ka, kd and KD from these studies are shown in Table 7. Most of the mutations that result in improved binding are located outside of the CDRs. In particular, in these studies, a single R71W substitution present in DART-2 (anti-ROR1-VL(2)) increased binding by more than two fold.

為了進一步表徵變異的抗-ROR1-VL和抗-ROR1-VH結構域，使用兩種不同的細胞毒性T淋巴細胞(CTL)測定來評估幾種ROR1×CD3雙抗體介導重定向細胞殺傷的能力。在一個測定中，將ROR1×CD3雙特異性雙抗體或陰性對照雙抗體(缺少ROR1-結合位點)與全T-細胞和靶腫瘤細胞一起孵育，並通過測量由損傷的細胞向培養基中的乳酸脫氫酶(LDH)的釋放來測定細胞毒性百分比(即細胞殺傷)。使用定量測量LDH釋放的CytoTox 96®非放射性細胞毒性測定試劑盒(Promega)進行這些測定，基本上如下所述。使用在測定培養基(不含酚紅的RPMI 1640、10％FBS、1％青黴素/鏈黴素)中密度為4×105個細胞/mL和在測定開始時生存率高於90％的靶細胞(例如腫瘤靶細胞)，以及以適當的密度懸浮在測定培養基中以實現10:1的效應子與靶標(E:T)細胞比(或期望的E:T比)的、分離的純化的人T-細胞。50μL靶細胞懸浮物(〜20,000個細胞)、100μL效應細胞懸浮物(對於10:1的 E:T比例為200,000個細胞)和50μL連續稀釋的雙特異性ROR1×CD3雙抗體或陰性對照雙抗體(缺乏ROR1-結合位點)被加入微量滴定板的重複的孔(duplicate wells)中，並孵育(37℃，5％CO₂ ) 24小時。在孵育結束時，加入30μL裂解溶液，將板孵育10分鐘以完全裂解靶細胞。然後將板離心(212 × g，5分鐘)，將40μL上清液從測定板的每個孔轉移至平底ELISA板中，並將40μL LDH底物溶液加入每個孔中。將板在室溫，黑暗中孵育10-20分鐘，並加入40μL終止溶液(Promega Cat＃G183A)。在Victor2 Multilabel板閱讀器(Perkin Elmer＃1420-014)上，在1小時內，在490nm處測量光密度。使用下式從光密度(OD)資料計算特定的細胞裂解：並且使用GraphPad Prism 6軟體，通過將細胞毒性值曲線擬合到S形(sigmoidal)劑量-回應函數來生成劑量-回應曲線。To further characterize the mutated anti-ROR1-VL and anti-ROR1-VH domains, two different cytotoxic T lymphocyte (CTL) assays were used to assess the ability of several ROR1×CD3 diabody-mediated retargeting cell killing . In one assay, a ROR1×CD3 bispecific diabody or a negative control diabody (lacking a ROR1-binding site) is incubated with whole T-cells and target tumor cells and measured by the damaged cells into the culture medium. The release of lactate dehydrogenase (LDH) is used to determine the percentage of cytotoxicity (ie, cell killing). These assays were performed using a CytoTox 96® non-radioactive cytotoxicity assay kit (Promega) that quantitatively measures LDH release, essentially as described below. Target cells with a density of 4 x 105 cells/mL in the assay medium (RPMI 1640, 10% FBS, 1% penicillin/streptomycin without phenol red) and a survival rate above 90% at the start of the assay ( For example, tumor target cells), and isolated purified human T suspended in assay medium at an appropriate density to achieve a 10:1 effector to target (E:T) cell ratio (or desired E:T ratio) -cell. 50 μL of target cell suspension (~20,000 cells), 100 μL of effect cell suspension (200:000 cells for a 10:1 E:T ratio) and 50 μL of serially diluted bispecific ROR1×CD3 diabody or negative control diabody (The lack of ROR1-binding sites) was added to duplicate wells of microtiter plates and incubated (37 ° C, 5% CO ₂ ) for 24 hours. At the end of the incubation, 30 μL of the lysis solution was added and the plate was incubated for 10 minutes to completely lyse the target cells. The plates were then centrifuged (212 x g, 5 minutes), 40 [mu]L of supernatant was transferred from each well of the assay plate to a flat bottom ELISA plate, and 40 [mu]L of LDH substrate solution was added to each well. The plates were incubated for 10-20 minutes at room temperature in the dark and 40 [mu]L of stop solution (Promega Cat# G183A) was added. The optical density was measured at 490 nm over 1 hour on a Victor2 Multilabel plate reader (Perkin Elmer #1420-014). Calculate specific cell lysis from optical density (OD) data using the following formula: And using the GraphPad Prism 6 software, a dose-response curve was generated by fitting a cytotoxicity curve to a sigmoidal dose-response function.

在另一種測定中，將ROR1 × CD3雙特異性雙抗體或陰性對照雙抗體(缺乏ROR1-結合位點)與全 T-細胞和靶JIMT-1細胞(其已經被工程化以表達螢光素酶(luc)報導基因(JIMT-1-Luc細胞))一起孵育，並通過測量靶細胞的細胞螢光素酶活性的發光(LUM)測定來測定細胞毒性。這些測定的製備和設置基本上與上述LDH測定相同。孵育後，從每個孔中取出100μL孵育基，隨後向每個孔中加入100μL Steady-Glo螢光素酶底物，然後向上/向下吹吸數次以完全裂解靶細胞。將板在室溫，黑暗中孵育10分鐘，然後使用VictorX4 Multilabel板閱讀器(Perkin Elmer＃1420-014)測量發光強度，以發光相對光單位(RLU)作為讀出。RLU指示靶細胞的相對存活力。使用GraphPad Prism 6軟體通過將RLU值曲線擬合到S形劑量-回應函數來生成劑量-回應曲線。In another assay, a ROR1 × CD3 bispecific diabody or a negative control diabody (lacking a ROR1-binding site) with whole T-cells and target JIMT-1 cells (which have been engineered to express luciferin) The enzyme (luc) reporter gene (JIMT-1-Luc cells) was incubated together and cytotoxicity was determined by a luminescence (LUM) assay measuring the luciferase activity of the target cells. The preparation and setup of these assays were essentially the same as the LDH assay described above. After the incubation, 100 μL of the incubation group was taken out from each well, and then 100 μL of Steady-Glo luciferase substrate was added to each well, and then pipetted up/down several times to completely lyse the target cells. The plates were incubated for 10 minutes at room temperature in the dark, and then the luminescence intensity was measured using a VictorX4 Multilabel plate reader (Perkin Elmer #1420-014), with luminescence relative light units (RLU) as readout. RLU indicates the relative viability of target cells. A dose-response curve was generated by fitting the RLU value curve to a sigmoidal dose-response function using GraphPad Prism 6 software.

對於這些研究，使用JIMT-1乳腺癌細胞、HBL-2套細胞淋巴瘤細胞或Jeko-1套細胞淋巴瘤細胞作為腫瘤靶細胞，並且使用雙抗體(DART-1、DART-2、DART-14、DART-15、DART-16、DART-20、DART-22、DART-23和DART-25)的五倍連續稀釋物。代表性的細胞毒性曲線示於圖 8A-8B 、9A-9B 和10A-10C 中。圖 10A-10C 中的曲線的EC50和最大回應值在表 8 中提供。這些研究證明，包括優化的抗-ROR1-VL和/或VH結構域的雙抗體(例如DART-2、DART-8、DART-20、DART-22、DART-23和DART-25)相對於具有親本抗-ROR1-VL和/或VH結構域的雙抗體表現介導對腫瘤細胞的重定向細胞殺傷的更好的能力。尤其地，相比DART-1，對ROR1具有更高的親和力的雙抗體和那些在VH結構域中包括A93T的雙抗體顯示介導重定向細胞殺傷的增強的能力。DART-23和DART-25的EC50值是DART-1的二十分之一到十分之一。 For these studies, JIMT-1 breast cancer cells, HBL-2 mantle cell lymphoma cells, or Jeko-1 mantle cell lymphoma cells were used as tumor target cells, and diabody (DART-1, DART-2, DART-14) was used. Five-fold serial dilutions of DART-15, DART-16, DART-20, DART-22, DART-23, and DART-25). Representative cytotoxicity curves are shown in Figures 8A-8B , 9A-9B and 10A-10C . The EC50 and maximum response values for the curves in Figures 10A-10C are provided in Table 8 . These studies demonstrate that dual antibodies (eg, DART-2, DART-8, DART-20, DART-22, DART-23, and DART-25), including optimized anti-ROR1-VL and/or VH domains, have relative The diabody of the parental anti-ROR1-VL and/or VH domains is shown to mediate a better ability to redirect cell killing of tumor cells. In particular, diabodies with higher affinity for ROR1 and those with A93T in the VH domain showed enhanced ability to mediate redirection of cell killing compared to DART-1. The EC50 values for DART-23 and DART-25 are between one-twentieth and one-tenth of DART-1.

實施例Example 22

抗anti- -ROR1-ROR1 可變結構域的進一步優化和雙特異性三鏈雙抗體的生成Further optimization of variable domains and generation of bispecific triplex diabody

為了進一步優化抗-ROR1-VL和抗-ROR1-VH結構域，將幾種改變引入到抗-ROR1-可變結構域中，以降低免疫原性。修飾親本抗-ROR1-VL結構域和優化的抗ROR1-VL(2)結構域以除去存在於Kabat位置63和64(對應於SEQ ID NO:6 和SEQ ID NO:11 的位置67)之間的額外的甘氨酸(G)殘基。被命名為“抗 -ROR1-VL(1) ”和“抗 -ROR1-VL(14) ”(分別為SEQ ID NO:10 和SEQ ID NO:23 ，也見上述表 6 )的所得抗-ROR1-VL結構域，被併入到具有兩條或三條多肽鏈的ROR1×CD3雙特異性雙抗體中，並與不同的抗-ROR1-VH結構域配對，如下文更詳細描述。To further optimize the anti-ROR1-VL and anti-ROR1-VH domains, several alterations were introduced into the anti-ROR1-variable domain to reduce immunogenicity. The parental anti-ROR1-VL domain and the optimized anti-ROR1-VL(2) domain are modified to remove the presence of the Kabat positions 63 and 64 (corresponding to position 67 of SEQ ID NO: 6 and SEQ ID NO: 11 ) Additional glycine (G) residues between. The resulting anti- ROR1, designated " anti- ROR1-VL(1) " and " anti- ROR1-VL(14) " ( SEQ ID NO: 10 and SEQ ID NO: 23 , respectively, see also Table 6 above) The -VL domain, which is incorporated into a ROR1 x CD3 bispecific diabody with two or three polypeptide chains, is paired with a different anti-ROR1-VH domain, as described in more detail below.

修飾此類分子的抗-ROR1-VH結構域以除去存在於CDR_H 1和CDR_H 2中的兩個混雜的高親和性MHC II類結合序列。具體地，將Kabat 位置37(對應於SEQ ID NO:7 的位置37)處的纈氨酸突變為異亮氨酸(“V37I ”)，以破壞CDR_H 1中存在的免疫原性序列，和將Kabat 位置63處的纈氨酸(對應於SEQ ID NO:7 的位置64)突變為丙氨酸(“V63A ”)，以破壞CDR_H 2中存在的免疫原性序列。命名為“抗 -ROR1-VH(8) ”(SEQ ID NO:31 ，見上表 6 )的所得VH結構域被併入到具有兩條或三條鏈的ROR1×CD3雙特異性雙抗體中，如下文更詳細描述。The anti-ROR1-VH domain of such a molecule is modified to remove two promiscuous high affinity MHC class II binding sequences present in CDR _H1 and CDR _H2 . Specifically, the proline at Kabat position 37 (corresponding to position 37 of SEQ ID NO: 7 ) is mutated to isoleucine (" V37I ") to disrupt the immunogenic sequence present in CDR _H1 , and The proline at position 63 of Kabat (corresponding to position 64 of SEQ ID NO: 7 ) was mutated to alanine (" V63A ") to disrupt the immunogenic sequence present in CDR _H2 . The resulting VH domain designated " Anti- ROR1-VH(8) " ( SEQ ID NO: 31 , see Table 6 above) was incorporated into a ROR1 x CD3 bispecific diabodies with two or three strands, As described in more detail below.

生成了具有兩條鏈的兩種ROR1 x CD3雙特異性雙抗體，其包括抗-ROR1-VL(14)。這些雙抗體被命名為：“DART-32 ”，其包括抗-ROR1-VL(14)和抗-ROR1-VH(7)；和“DART-33 ”，其包括抗-ROR1-VL(14)和抗-ROR1-VH(8)(見上表 7 )。上文詳細提供了這些示例性ROR1×CD3雙特異性雙鏈雙抗體的第一和第二多肽鏈的一般結構。Two ROR1 x CD3 bispecific diabodies with two strands were generated, including anti-ROR1-VL (14). These diabodyes are named: " DART-32 ", which includes anti-ROR1-VL (14) and anti-ROR1-VH (7); and " DART-33 ", which includes anti-ROR1-VL (14) And anti-ROR1-VH(8) (see Table 7 above). The general structure of the first and second polypeptide chains of these exemplary ROR1 x CD3 bispecific double-chain diabody is provided in detail above.

DART-32DART-32

DART-32的第一多肽鏈的氨基酸序列(SEQ ID NO:114 )如下所示(抗-ROR1-VL(14)以底線表示)： QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFSGSS SGADWYLTIS SLQSEDEADY YCGTDYPGNY LFGGGTQLTV LG GGGSGGGG EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMNWVRQA PGKGLEWVGR IRSKYNNYAT YYADSVKGRF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVS WFAYWGQGTL VTVSSGGCGG GKVAALKEKV AALKEKVAAL KEKVAALKEThe amino acid sequence of the first polypeptide chain of DART-32 ( SEQ ID NO: 114 ) is shown below (anti-ROR1-VL (14) is indicated by the bottom line): QLVLTQSPSA SASLGSSVKL TCTLSSGHKT DTIDWYQQQP GKAPRYLMKL EGSGSYNKGS GVPDRFSGSS SGADWYLTIS SLQSEDEADY YCGTDYPGNY LFGGGTQLTV LG GGGSGGGG EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMNWVRQA PGKGLEWVGR IRSKYNNYAT YYADSVKGRF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVS WFAYWGQGTL VTVSSGGCGG GKVAALKEKV AALKEKVAAL KEKVAALKE

DART-32的第二多肽鏈的氨基酸序列與上文提供的DART-25的第二多肽鏈(SEQ ID NO:97 )一致。The amino acid sequence of the second polypeptide chain of DART-32 is identical to the second polypeptide chain of DART-25 provided above ( SEQ ID NO: 97 ).

DART-33DART-33

DART-33的第一多肽鏈的氨基酸序列與上文提供的DART-32的第一多肽鏈(SEQ ID NO:114 )一致。The amino acid sequence of the first polypeptide chain of DART-33 is identical to the first polypeptide chain of DART-32 provided above ( SEQ ID NO: 114 ).

DART-33的第二多肽鏈的氨基酸序列(SEQ ID NO:115 )如下所示(抗-ROR1-VH(8)以底線表示)： QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGG QE QLVESGGGLV QPGGSLRLSC AASGFTFSDY YMSWIRQAPG KGLEWVATIY PSSGKTYYAD SAKGRLTISS DNAKDSLYLQ MNSLRAEDTA VYYCTRDSYA DDAALFDIWG QGTTVTVSS G GCGGGEVAAL EKEVAALEKE VAALEKEVAA LEKThe amino acid sequence of the second polypeptide chain of DART-33 ( SEQ ID NO: 115 ) is shown below (anti-ROR1-VH(8) is indicated by the bottom line): QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGG QE QLVESGGGLV QPGGSLRLSC AASGFTFSDY YMSWIRQAPG KGLEWVATIY PSSGKTYYAD SAKGRLTISS DNAKDSLYLQ MNSLRAEDTA VYYCTRDSYA DDAALFDIWG QGTTVTVSS G GCGGGEVAAL EKEVAALEKE VAALEKEVAA LEK

此外，生成了具有三條鏈並具有Fc區域的四種ROR1×CD3雙特異性雙抗體，並命名為：“DART-A ”，其包括親本抗-ROR1-VL(SEQ ID NO:6 )和抗-ROR1-VH(SEQ ID NO:7 )結構域；“DART-B ”，其包括抗-ROR1-VL(1) (SEQ ID NO:10 )和親本抗-ROR1-VH(SEQ ID NO:7 )結構域；“DART-C ”，其包括抗-ROR1-VL(14) (SEQ ID NO:23 )和抗-ROR1-VH(7) (SEQ ID NO:30 )；和“DART-D ”，其包括抗-ROR1-VL(14) (SEQ ID NO:23 )和抗-ROR1-VH(8) (SEQ ID NO:31 )。上文詳細提供了這些示例性ROR1×CD3雙特異性三鏈雙抗體的第一、第二和第三多肽鏈的一般結構和氨基酸序列。在DART-A、DART-B、DART-C和DART-D中存在的具體抗-ROR1-VL和抗-ROR1-VH結構域提供於表 9 中。 -：不確定In addition, four ROR1 x CD3 bispecific diabodies with three strands and having an Fc region were generated and designated: " DART-A ", which includes the parental anti-ROR1-VL ( SEQ ID NO: 6 ) and anti-ROR1-VH ( SEQ ID NO:7 ) domain; " DART-B ", which includes anti-ROR1-VL(1) ( SEQ ID NO: 10 ) and parental anti-ROR1-VH ( SEQ ID NO) :7 ) a domain; " DART-C " comprising anti-ROR1-VL (14) ( SEQ ID NO: 23 ) and anti-ROR1-VH (7) ( SEQ ID NO: 30 ); and " DART- D ", which includes anti-ROR1-VL (14) ( SEQ ID NO: 23 ) and anti-ROR1-VH (8) ( SEQ ID NO: 31 ). The general structure and amino acid sequence of the first, second and third polypeptide chains of these exemplary ROR1 x CD3 bispecific triplex diabodyes are provided in detail above. The specific anti-ROR1-VL and anti-ROR1-VH domains present in DART-A, DART-B, DART-C and DART-D are provided in Table 9 . -:uncertain

通過夾心ELISA檢驗雙特異性ROR1x CD3二鏈和三鏈雙抗體DART-1和DART-A結合ROR1和CD3兩者的能力。簡言之，用shROR1-His塗覆微量滴定板，洗滌板，並用DART-1或DART-A的三倍連續稀釋物孵育。使用通過鏈酶抗生物素-HRP檢測的生物素化的CD3評估與固定化的ROR1結合的雙抗體的量。在板閱讀器上分析所有樣品，並生成結合曲線。在另外的研究中，基本上如上所述檢驗雙特異性ROR1x CD3三鏈雙抗體DART-A、DART-C和DART-D結合ROR1和CD3兩者的能力。來自這些研究的結合曲線(圖 11A-11B )證明二鏈和三鏈雙抗體都能夠進行雙重抗原結合，並且在具有優化的抗-ROR1-VL和抗-ROR1-VH結構域的三鏈雙抗體中雙重抗原結合被保留。通過FACS分析評價DART-D結合至三種表達ROR1的癌細胞系(HOP-92、PC-3和HBL-2)和表達CD3的人原代T-細胞的表面的能力。簡言之，將細胞(100μL中的0.5至1.0×10⁶ 個細胞/ mL)與0.12nM-10nM DART-D(在含有10％人AB血清的FACS緩衝液中，100μL終體積)在微量滴定板中孵育20- 60分鐘。細胞被洗滌兩次，將細胞與識別E-螺旋/K-螺旋(EK)異源二聚化區域的生物素綴合的小鼠抗EK-螺旋抗體(100μL的1μg/mL與1:500稀釋的鏈黴抗生物素-藻紅素混合)一起孵育45分鐘。然後洗滌細胞並用FACS緩衝液重懸，並用BD FCS Canto II流式細胞儀，使用FlowJo v10軟體進行分析。如圖 12A-12D 所示，DART-D結合表達ROR1的人癌細胞(圖 12A-12C )和結合表達CD3的 T細胞(圖 12D )。The ability of the bispecific ROR1x CD3 di-chain and triple-stranded diabody DART-1 and DART-A to bind both ROR1 and CD3 was tested by sandwich ELISA. Briefly, microtiter plates were coated with shROR1-His, plates were washed and incubated with three-fold serial dilutions of DART-1 or DART-A. The amount of diabody bound to immobilized ROR1 was assessed using biotinylated CD3 detected by streptavidin-HRP. All samples were analyzed on a plate reader and a binding curve was generated. In a separate study, the ability of the bispecific ROR1x CD3 triplex diabody DART-A, DART-C and DART-D to bind both ROR1 and CD3 was tested essentially as described above. Binding curves from these studies ( Figures 11A-11B ) demonstrate that both double-stranded and triple-stranded diabody are capable of dual antigen binding and that triple-stranded diabodies with optimized anti-ROR1-VL and anti-ROR1-VH domains The double antigen binding is retained. The ability of DART-D to bind to the surface of three ROR1-expressing cancer cell lines (HOP-92, PC-3 and HBL-2) and human primary T-cells expressing CD3 was evaluated by FACS analysis. Briefly, cells (0.5 to 1.0×10 ⁶ cells/mL in 100 μL) and 0.12 nM-10 nM DART-D (in FACS buffer containing 10% human AB serum, 100 μL final volume) in microtiter titration Incubate in the plate for 20-60 minutes. The cells were washed twice and the cells were conjugated to a biotin-conjugated mouse anti-EK-helical antibody (100 μL of 1 μg/mL and 1:500 diluted with biotin) that recognizes the E-helix/K-helix (EK) heterodimerization region. The streptavidin-phycoerythrin mixture was incubated for 45 minutes. The cells were then washed and resuspended in FACS buffer and analyzed using a BD FCS Canto II flow cytometer using FlowJo v10 software. As shown in FIGS. 12A-12D, DART-D ROR1 binding T cells expressing human cancer cells (FIGS. 12A-12C), and expression of binding to CD3 (FIG. 12D).

使用Biacore分析研究DART-32和DART-33的結合動力學，其中使shROR1-His在固定化的雙抗體上通過，如實施例 1 所述。從這些研究計算的k_a 、k_d 和K_D 在上表 7 中示出。Binding kinetics of DART-32 and DART-33 were studied using Biacore analysis in which shROR1-His was passed over the immobilized diabody as described in Example 1 . The k _a , k _d and K _D calculated from these studies are shown in Table 7 above.

使用Biacore分析研究DART-1、DART-A、DART-B和DART-C的結合親和力，其中使每種雙抗體構建體在固定化的ROR1上通過。簡言之，將shROR1-His捕獲在固定化的抗-PentaHis表面上，並與6.25-100nM的DART-1、DART-A、DART-B或DART-C一起孵育，通過Biacore分析測定結合的動力學。從這些研究計算的k_a 、k_d 和K_D 在上表 8 中示出。Binding affinities of DART-1, DART-A, DART-B and DART-C were studied using Biacore analysis, wherein each diabody construct was passed over immobilized ROR1. Briefly, shROR1-His was captured on immobilized anti-PentaHis surface and incubated with 6.25-100 nM of DART-1, DART-A, DART-B or DART-C, and binding kinetics were determined by Biacore analysis. learn. The k _a , k _d and K _D calculated from these studies are shown in Table 8 above.

這些研究證明，三鏈雙抗體例如DART-1的結合親和力與包括相同VL和VH的雙鏈雙抗體例如DART-A(見表 7 )的結合親和力相當。DART-25和DART-32的結合親和力幾乎與DART-A和DART-B的結合親和力相同，表明缺失額外的G殘基不改變結合親和力(見表 7 和表 8 )。此外，與包括親本VL和抗-ROR1-VH結構域的相應雙抗體(DART-1和DART-A)相比，DART-32和DART-C的結合增強超過兩倍(見表 7 和表 8 )。此外，DART-32和DART-33具有幾乎相同的結合親和力(見表 7 )，表明在CDR_H 1附近和CDR_H 2內引入去免疫化突變對結合親和力沒有負面影響。These studies demonstrate that the three chain diabody e.g. DART-1 binding affinity for double-stranded diabody comprises VH and VL, for example, the same DART-A (see Table 7) rather binding affinity. DART-25 binding affinity, and almost DART-32 binding affinity DART-A, and the same DART-B, indicating the absence of an additional G residue does not alter binding affinity (see Tables 7 and 8). Furthermore, compared to the corresponding diabody (DART-1 and the DART-A) and comprising a parent VL anti -ROR1-VH domains, binding DART-32 and the DART-C enhanced more than twice (see Table 7 and Table 8 ). Further, DART-32 and the DART-33 have almost the same binding affinity (see Table 7), suggesting that the introduction in the vicinity of CDR _H 1 and CDR _H 2 deimmunized mutation does not adversely affect the binding affinity.

實施例Example 33

細胞毒性研究Cytotoxicity study

基本上如實施例 1 中所述，使用LDH釋放測定法來評價雙特異性ROR1xCD3雙鏈和三鏈雙抗體DART-1和DART-A介導重定向細胞殺傷的能力。對於這些研究，將ROR1 x CD3雙特異性雙抗體或陰性對照雙抗體(缺乏ROR1-結合位點)與效應子全T-細胞和靶腫瘤細胞(JIMT-1乳腺癌細胞、A549肺癌細胞、HBL-2套細胞淋巴瘤細胞)以10:1的效應子與靶的比例一起孵育24小時。在其他研究中，效應子PBMC細胞和靶RECA0201癌幹細胞以30:1的效應子與靶的比例被使用。使用DART-1、DART-A和陰性對照的5倍連續稀釋物。每種靶腫瘤細胞類型的代表性細胞毒性曲線示於圖 13A-13D 中。在進一步的研究中，使用實施例1中描述的LDH釋放測定法評估雙特異性ROR1 x CD3三鏈雙抗體DART-A、DART-C和DART-D介導細胞毒性的能力。對於這些研究，將ROR1 x CD3雙特異性雙抗體或陰性對照(缺乏ROR1-結合位點)與效應子全T-細胞和靶腫瘤細胞(JIMT-1乳腺癌細胞、NCI-H1957細胞)以10:1的效應子與靶的比例一起孵育24小時。使用DART-A、DART-C、DART-D和陰性對照的5倍連續稀釋物。每種靶腫瘤細胞類型的代表性細胞毒性曲線示於圖 14A-14B 中。在沒有效應細胞的情況下沒有觀察到細胞殺傷。這些研究表明，三鏈雙抗體保留介導細胞毒性的能力，並且具有優化的抗-ROR1-VL和抗-ROR1-VH結構域的三鏈雙抗體保留在雙鏈形式中觀察到的增強的介導重定向細胞殺傷的能力。The LDH release assay was used essentially as described in Example 1 to assess the ability of the bispecific ROR1xCD3 double-stranded and triple-stranded diabody DART-1 and DART-A to mediate redirect cell killing. For these studies, ROR1 x CD3 bispecific diabody or negative control diabody (lack of ROR1-binding site) and effector whole T-cells and target tumor cells (JIMT-1 breast cancer cells, A549 lung cancer cells, HBL) - 2 sets of cell lymphoma cells were incubated with a 10:1 effector to target ratio for 24 hours. In other studies, effector PBMC cells and target RECA0201 cancer stem cells were used at a ratio of 30:1 effector to target. A 5-fold serial dilution of DART-1, DART-A and a negative control was used. Representative cytotoxicity curves for each target tumor cell type are shown in Figures 13A-13D . In a further study, the ability of the bispecific ROR1 x CD3 triplex diabody DART-A, DART-C and DART-D to mediate cytotoxicity was assessed using the LDH release assay described in Example 1. For these studies, ROR1 x CD3 bispecific diabody or negative control (lack of ROR1-binding site) and effector whole T-cell and target tumor cells (JIMT-1 breast cancer cells, NCI-H1957 cells) were 10 The effector of :1 was incubated with the target ratio for 24 hours. Five-fold serial dilutions of DART-A, DART-C, DART-D and negative controls were used. Representative cytotoxicity curves for each target tumor cell type are shown in Figures 14A-14B . No cell killing was observed in the absence of effector cells. These studies indicate that triple-stranded diabody retains the ability to mediate cytotoxicity, and that the triple-stranded diabody with optimized anti-ROR1-VL and anti-ROR1-VH domains retains the enhanced mediators observed in the double-stranded form. Guides the ability to redirect cells to kill.

在另外的研究中，使用另外的靶腫瘤細胞類型：HBL-2 B-細胞淋巴瘤細胞；HOP-92肺腺癌細胞；PC-3M***癌細胞；Daoy成神經管細胞瘤細胞；和Saos-2、U-2 OS和MG-63骨肉瘤細胞來評價代表性雙特異性ROR1 × CD3三鏈雙抗體(DART-D；5倍連續稀釋物)的細胞毒性活性。CHO細胞也包括在這些研究中作為ROR1陰性對照靶細胞。對於這些研究，在單獨的實驗中使用來自不同供體的原代T-細胞。針對不同的靶細胞系，有時使用原代T-細胞不同供體。對於每種細胞系測試的供體數目如下：MG-63(2個供體)，Saos-2(5個供體)，U2-OS(2個供體)，HBL-2 (3個供體)，HOP-92 (5個供體)、Daoy(3個供體)和PC-3 (7個供體)。對於每種靶細胞類型，T-細胞來自代表性供體的劑量依賴性殺傷曲線顯示於圖 15A-15H 。在評價的7種靶細胞系中觀察到範圍為0.0013-0.056nM的EC₅₀ 值(在每個圖中的括弧中示出)，其中HBL-2是最敏感的細胞系(EC₅₀ =0.0013ng/mL)。在評價的最高濃度(10,000ng/mL)，用對照DART分子觀察到最小或沒有活性。在ROR1-陰性CHO細胞中，在DART-D存在的情況下沒有觀察到細胞毒性，證實雙特異性ROR1 x CD3雙抗體對表達ROR1的靶細胞的活性的特異性。這些研究進一步證實，雙特異性ROR1 x CD3三鏈雙抗體(例如，DART-D)介導表達ROR1的靶細胞的有效的，特異性的重定向殺傷。In additional studies, additional target tumor cell types were used: HBL-2 B-cell lymphoma cells; HOP-92 lung adenocarcinoma cells; PC-3M prostate cancer cells; Daoy medulloblastoma cells; and Saos- 2. U-2 OS and MG-63 osteosarcoma cells were used to evaluate the cytotoxic activity of a representative bispecific ROR1 x CD3 triple-chain diabody (DART-D; 5-fold serial dilution). CHO cells were also included as ROR1 negative control target cells in these studies. For these studies, primary T-cells from different donors were used in separate experiments. Different donors of primary T-cells are sometimes used for different target cell lines. The number of donors tested for each cell line was as follows: MG-63 (2 donors), Saos-2 (5 donors), U2-OS (2 donors), HBL-2 (3 donors) ), HOP-92 (5 donors), Daoy (3 donors), and PC-3 (7 donors). The dose-dependent killing curve of T-cells from representative donors for each target cell type is shown in Figures 15A-15H . Evaluation was observed in the 7 cell lines to the target range of EC ₅₀ values 0.0013-0.056nM (each figure in parentheses is shown), wherein HBL-2 was the most sensitive cell lines (EC ₅₀ = 0.0013ng /mL). At the highest concentration evaluated (10,000 ng/mL), minimal or no activity was observed with control DART molecules. In ROR1-negative CHO cells, no cytotoxicity was observed in the presence of DART-D, confirming the specificity of the bispecific ROR1 x CD3 diabody for the activity of target cells expressing ROR1. These studies further confirmed that the bispecific ROR1 x CD3 triplex diabody (eg, DART-D) mediates efficient, specific redirected killing of target cells expressing ROR1.

在人PBMC中，單獨地或在以10:1的E:T細胞比存在表達ROR1的靶細胞(NCI-H1975肺癌細胞)的情況下，通過FACS評估由代表性雙特異性ROR1 x CD3三鏈雙抗體(DART-D)誘導的T-細胞啟動水準。簡言之，將PBMC (100-150μL測定培養基(RPMI 1640 + 10％FBS)中的200,000個細胞/孔)，單獨地或與靶細胞(在50μL中的20,000個細胞/孔)一起，與指示濃度的DART-D的連續稀釋物在微量滴定板的重複的孔中一起在37℃孵育24小時。如上詳細描述地，將來自每個孔的40μL上清液用於LDH釋放測量，剩餘的上清液用於測量細胞因數。簡言之，在測定板中用FACS緩衝液(100μL/孔)中的CD8-FITC、CD4-APC、CD25-PE和CD69-PECy5抗體(BD Biosciences)標記細胞。將板(在4℃的黑暗中)孵育30分鐘。然後洗滌細胞並重懸於FACS緩衝液中，並基本上如上文實施例2所述進行分析。此外，根據製造商的說明使用BD CBA 人Th1/Th2細胞因數試劑盒，在從相同實驗收集的培養物上清液中測量IFN-γ、IL-2、IL-4、IL-6、IL-10和TNF-α的細胞因數水準。使用FCAP Array(v3.0.1，BD Biosciences)測定細胞因數濃度。使用樣品強度值從4-參數標準曲線外推標準物濃度範圍(0-5000pg/mL)之外的值。這些研究的結果顯示在圖 16A-16B 、17A-17D 和18A-18E 中。In human PBMC, representative bispecific ROR1 x CD3 triple strands were assessed by FACS, either alone or in the presence of a target cell expressing ROR1 (NCI-H1975 lung cancer cells) at a ratio of 10:1 E:T cells. Double antibody (DART-D) induced T-cell activation level. Briefly, PBMC (200,000 cells/well in 100-150 μL assay medium (RPMI 1640 + 10% FBS), either alone or with target cells (20,000 cells/well in 50 μL), with instructions Serial dilutions of DART-D were incubated together in replicate wells of microtiter plates for 24 hours at 37 °C. As described in detail above, 40 [mu]L of supernatant from each well was used for LDH release measurements and the remaining supernatant was used to measure cytokines. Briefly, cells were labeled with CD8-FITC, CD4-APC, CD25-PE and CD69-PECy5 antibodies (BD Biosciences) in FACS buffer (100 μL/well) in assay plates. The plates (in the dark at 4 ° C) were incubated for 30 minutes. The cells were then washed and resuspended in FACS buffer and analyzed essentially as described in Example 2 above. In addition, IFN-γ, IL-2, IL-4, IL-6, IL- were measured in culture supernatants collected from the same experiment using the BD CBA Human Th1/Th2 cytokine kit according to the manufacturer's instructions. 10 and cytokine levels of TNF-α. Cytokine concentrations were determined using a FCAP Array (v3.0.1, BD Biosciences). Values other than the standard concentration range (0-5000 pg/mL) were extrapolated from the 4-parameter standard curve using sample intensity values. The results of these studies are shown in Figures 16A-16B , 17A-17D and 18A-18E .

DART-D介導的T-細胞啟動與靶細胞的細胞毒性相關(圖 16A-16B )。在所有評價的濃度，在靶細胞存在的情況下觀察到顯著的DART-D介導的細胞毒性(圖 16A )。相比之下，在CTL測定中，當單獨的PBMC與DART-D或對照DART孵育時，沒有觀察到細胞毒性(圖 16B )。流式細胞術分析顯示，在表達ROR1的靶細胞存在的情況下，通過DART-D，CD4⁺ (圖 17A 和 17C )和CD8⁺ T-細胞亞群(圖 17B 和 17D )上的CD69(圖 17A-17B )和CD25(圖 17C-17D )T細胞啟動標記物的劑量依賴性方式的上調。這些資料表明，由本發明的雙特異性ROR1 x CD3雙抗體介導的T細胞啟動依賴於效應細胞-靶細胞共接合(co-engagement)。與T-細胞啟動標記物一致，當在存在表達ROR1的靶細胞(實心符號)的情況下用DART-D處理PBMC時，觀察到測量的所有6種細胞因數的水準的劑量依賴性增加(IFN-γ、TNF-α、IL-10、IL-6、IL-4和IL-2，分別示於圖 18A-18F )。然而，當單獨的PBMC用DART-D或對照陰性對照雙抗體(空心符號)處理時，沒有觀察到細胞因數釋放。DART-D mediated T-cell initiation is associated with cytotoxicity of target cells ( Figures 16A-16B ). Significant DART-D mediated cytotoxicity was observed in the presence of target cells at all concentrations evaluated ( Figure 16A ). In contrast, in the CTL assay, no cytotoxicity was observed when PBMC alone was incubated with DART-D or control DART ( Fig. 16B ). Flow cytometry analysis showed that, in the case of the presence of target cells expressing ROR1 by DART-D, CD69 on CD4 ⁺ (FIGS. 17A and 17C) and CD8 ⁺ T- cell subsets (FIGS. 17B and 17D) (FIG. Up-regulation of the dose-dependent manner of 17A-17B ) and CD25 ( Fig. 17C-17D ) T cell initiation markers. These data indicate that T cell initiation mediated by the bispecific ROR1 x CD3 diabody of the invention is dependent on effector-target cell co-engagement. Consistent with the T-cell initiation marker, a dose-dependent increase in the level of all six cytokines measured was observed when PBMCs were treated with DART-D in the presence of target cells expressing ROR1 (closed symbols) (IFN) - γ, TNF-α, IL-10, IL-6, IL-4 and IL-2 are shown in Figures 18A-18F , respectively. However, no cytokine release was observed when PBMC alone was treated with DART-D or a control negative control diabody (open symbols).

實施例Example 44

體內研究In vivo research

在幾種癌症模型中檢驗了雙特異性ROR1 x CD3雙鏈和三鏈雙抗體的體內活性。在共混HBL-2套細胞淋巴瘤模型中檢驗DART-1和DART-A的抗腫瘤活性。簡言之，將HBL-2套細胞淋巴瘤細胞(5×10⁶ )與啟動的人T-細胞以5:1的比例預混合，並於第0天皮下(SQ)植入到NOD/SCID(NOG)小鼠(8只雌性/組)中。從第0天開始，每天一次，通過靜脈內(IV)注射處理小鼠，達四天，在一項研究中用DART-1(0.004、0.02、0.1或1mg/kg)或僅僅媒介物，和在另一項研究中用DART-A(0.00016、0.0008、0.004或0.02mg/kg)或僅僅媒介物。在研究過程中監測腫瘤生長。這些實驗的結果(圖 19A-19B )顯示，DART-1和DART-A都能夠在這種鼠異種移植模型中預防或抑制腫瘤發展。The in vivo activities of bispecific ROR1 x CD3 double-stranded and triple-stranded diabody were tested in several cancer models. The anti-tumor activity of DART-1 and DART-A was tested in a blended HBL-2 mantle cell lymphoma model. Briefly, HBL-2 mantle cell lymphoma cells (5×10 ⁶ ) were premixed with activated human T-cells in a 5:1 ratio and implanted subcutaneously (SQ) onto NOD/SCID on day 0. (NOG) mice (8 females/group). From day 0, once daily, mice were treated by intravenous (IV) injection for up to four days, using DART-1 (0.004, 0.02, 0.1 or 1 mg/kg) or vehicle alone in one study, and In another study DART-A (0.00016, 0.0008, 0.004 or 0.02 mg/kg) or vehicle alone. Tumor growth was monitored during the course of the study. The results of these experiments ( Figures 19A-19B ) show that both DART-1 and DART-A are able to prevent or inhibit tumor progression in this murine xenograft model.

在進一步的研究中，在PBMC-重構的HOP-92肺腺癌模型中檢驗DART-A和DART-D的抗腫瘤活性。簡言之，將HOP-92細胞(5×10⁶ )重懸於50μL Ham's F12培養基中，與50μL 基質膠(Matrigel)組合，然後在研究第0天通過皮內(ID)注射植入MHCl1-/-小鼠(6-7只雌性/組)中，在研究第13天，通過腹膜內(IP)注射(200μL，Ham's F12培養基)植入人PBMC(1×10⁷ 個存活細胞)。在第26天，將動物隨機分組，並通過IV注射用DART-A (5、50或500μg/ kg)，DART-D (0.5、5、50或500μg/ kg)或僅僅媒介物處理，每7天一次，共5劑量。在研究過程中監測腫瘤體積。該實驗的結果(圖 20A-20B )顯示，DART-A和DART-D都能夠在這種鼠異種移植模型中預防或抑制腫瘤發展。In a further study, the anti-tumor activity of DART-A and DART-D was tested in a PBMC-reconstituted HOP-92 lung adenocarcinoma model. Briefly, HOP-92 cells (5 x 10 ⁶ ) were resuspended in 50 μL of Ham's F12 medium, combined with 50 μL Matrigel, and then implanted with MHCl1-by intradermal (ID) injection on study day 0. In mice (6-7 females/group), human PBMCs (1 × 10 ⁷ viable cells) were implanted by intraperitoneal (IP) injection (200 μL, Ham's F12 medium) on the 13th day of the study. On day 26, animals were randomized and treated with DART-A (5, 50 or 500 μg/kg), DART-D (0.5, 5, 50 or 500 μg/kg) or vehicle alone, IV injection. Once a day, a total of 5 doses. Tumor volume was monitored during the course of the study. The results of this experiment ( Figures 20A-20B ) show that both DART-A and DART-D are able to prevent or inhibit tumor progression in this murine xenograft model.

在進一步的研究中，在PBMC-重構的NCI-H1975肺癌模型中檢驗DART-B和DART-D的抗腫瘤活性。簡言之，將NCI-H1975細胞(5×10⁶ )重懸於50μL Ham's F12培養基中，與50μL基質膠組合，然後在研究第0天通過ID注射植入MHCl1-/-小鼠(6只雌性/組)中。在研究的第7天，通過IP注射(200μL，Ham's F12培養基)植入人PBMC(1×10⁷ 個存活細胞)。在第15天，將動物隨機分組，並通過IV注射用DART-B(0.5、5、50、或500μg/ kg)，DART-D(0.5、5、50或500μg/ kg)或僅僅媒介物處理，每7天一次，共2劑量。該實驗的結果(圖 21A-21B )顯示，DART-B和DART-D都能夠在這種鼠異種移植模型中預防或抑制腫瘤發展。In a further study, the anti-tumor activity of DART-B and DART-D was tested in a PBMC-reconstituted NCI-H1975 lung cancer model. Briefly, NCI-H1975 cells (5×10 ⁶ ) were resuspended in 50 μL of Ham's F12 medium, combined with 50 μL of Matrigel, and then MHCl1-/- mice were implanted by ID injection on Study Day 0 (6 Female / group). Day 7 of the study, by IP injection (200μL, Ham's F12 medium) implanted in a human PBMC (1 × 10 ⁷ viable cells). On day 15, animals were randomized and treated with DART-B (0.5, 5, 50, or 500 μg/kg), DART-D (0.5, 5, 50 or 500 μg/kg) or vehicle alone by IV injection. , every 7 days, a total of 2 doses. The results of this experiment ( Figures 21A-21B ) show that both DART-B and DART-D are able to prevent or inhibit tumor progression in this murine xenograft model.

在進一步研究中，在共混REC1套細胞癌(mantle cancer)模型中檢驗DART-B的抗腫瘤活性。簡言之，將REC1細胞(5×10⁶ )與啟動的人T細胞以5:1的比例預混合，並在第0天皮下(SQ)植入到NOD / SCID(NOG)小鼠(8只雌性/組)中。從第0天開始，通過靜脈內(IV)注射，用DART-B(0.5、5、50或500μg/ kg)或僅僅媒介物處理小鼠，每天一次，持續4天。在研究過程中監測腫瘤生長。該實驗的結果(圖 22 )顯示，DART-B能夠在這種鼠異種移植模型中預防或抑制腫瘤發展。In a further study, the anti-tumor activity of DART-B was tested in a blended REC1 mantle cancer model. Briefly, REC1 cells (5×10 ⁶ ) were pre-mixed with activated human T cells in a 5:1 ratio and implanted subcutaneously (SQ) on day 0 into NOD / SCID (NOG) mice (8 Only female/group). From day 0, mice were treated with DART-B (0.5, 5, 50 or 500 μg/kg) or vehicle alone, by intravenous (IV) injection, once daily for 4 days. Tumor growth was monitored during the course of the study. The results of this experiment ( Figure 22 ) show that DART-B is able to prevent or inhibit tumor progression in this murine xenograft model.

在進一步的研究中，在PBMC-重構的REC1套細胞癌模型中檢驗DART-D的抗腫瘤活性。簡言之，在研究第0天通過IP注射(200μL，Ham's F12培養基)植入人PBMC(1×10⁷ 個存活細胞)。將REC1細胞(5×10⁶ )重懸於50μL Ham's F12培養基中，與50 μL基質膠組合，然後在研究的第1天通過ID注射植入MHCl1-/-小鼠(8只雌性/組)中。在第13天，將動物隨機分組，並通過IV注射用DART-D(0.05、0.5、5、50、或500μg/ kg)或僅僅媒介物處理，每7天一次，共4劑量。該實驗的結果(圖 23 )顯示，DART-D能夠在這種鼠異種移植模型中預防或抑制腫瘤發展。In a further study, the anti-tumor activity of DART-D was tested in a PBMC-reconstituted REC1 set of cell carcinoma models. Briefly, the study day 0 by IP injection (200μL, Ham's F12 medium) implanted in a human PBMC (1 × 10 ⁷ viable cells). REC1 cells (5×10 ⁶ ) were resuspended in 50 μL of Ham's F12 medium, combined with 50 μL of Matrigel, and then MHCl1-/- mice (8 females/group) were implanted by ID injection on the first day of the study. in. On day 13, animals were randomized and treated with DART-D (0.05, 0.5, 5, 50, or 500 μg/kg) or vehicle alone by IV injection, once every 7 days for a total of 4 doses. The results of this experiment ( Figure 23 ) show that DART-D is able to prevent or inhibit tumor progression in this murine xenograft model.

在進一步研究中，在共混DAOY促結蒂組織增生的小腦成神經管細胞瘤模型中檢驗DART-D的抗腫瘤活性。簡言之，將DAOY細胞(5×10⁶ )與啟動的人T-細胞以5:1的比例預混合，並在第0天皮下(SQ)植入到NOG小鼠(7只雌性/組)中。從第0天開始，通過靜脈內(IV)注射用DART(0.005、0.05、0.5、5或50ng/kg)或僅僅媒介物處理小鼠，每天一次，持續四天。在研究過程中監測腫瘤生長。該實驗的結果(圖 24 )顯示，DART-D能夠在這種鼠異種移植模型中預防或抑制腫瘤發展。In a further study, the anti-tumor activity of DART-D was tested in a model of cerebellar medulloblastoma that was fused with DAOY to promote pedicle tissue hyperplasia. Briefly, DAOY cells (5×10 ⁶ ) were pre-mixed with activated human T-cells in a ratio of 5:1 and implanted into NOG mice subcutaneously (SQ) on day 0 (7 females/group) )in. From day 0, mice were treated with DART (0.005, 0.05, 0.5, 5 or 50 ng/kg) or vehicle alone by intravenous (IV) injection once daily for four days. Tumor growth was monitored during the course of the study. The results of this experiment ( Figure 24 ) show that DART-D is able to prevent or inhibit tumor progression in this murine xenograft model.

實施例Example 55

三特異性三價結合分子的生成Generation of trispecific trivalent binding molecules

生成四種三特異性ROR1 x CD3 x CD8三價結合分子，其各自具有對ROR1特異性的一個結合位點(包括親本和/或優化的抗-ROR1-VL和抗-ROR1-VH結構域)、對CD3特異性的一個結合位點(包括CD3 mAb 1(D65G)的VL和抗-ROR1-VH結構域)和對CD8特異性的一個結合位點(包括TRX2的VL和抗-ROR1-VH結構域)。具有三條多肽鏈並且包括親本抗-ROR1-VL和抗-ROR1-VH結構域的三價結合分子TRIDENT-A、具有四條多肽鏈並且包括親本抗-ROR1-VL和抗-ROR1 -VH結構域的TRIDENT-B、具有三條多肽鏈並且包括優化的抗-ROR1-VL(14)和抗-ROR1-VH(8)結構域的TRIDENT-C和具有四條多肽鏈並且包括優化的抗-ROR1-VL(14)和抗-ROR1-VH(8)結構域的TRIDENT-D已經在上文中被討論。上文詳細提供了這些三鏈和四鏈ROR1 x CD3 x CD8三價結合分子的多肽鏈的一般結構。存在於TRIDENT-A、TRIDENT-B、TRIDENT-C和TRIDENT-D中的具體抗-ROR1-VL和抗-ROR1-VH結構域提供於表 10 中。Generation of four trispecific ROR1 x CD3 x CD8 trivalent binding molecules each having a binding site specific for ROR1 (including parental and/or optimized anti-ROR1-VL and anti-ROR1-VH domains) a binding site specific for CD3 (including the VL and anti-ROR1-VH domains of CD3 mAb 1 (D65G)) and a binding site specific for CD8 (including VL and anti-ROR1- of TRX2) VH domain). Trivalent binding molecule TRIDENT-A having three polypeptide chains and including the parental anti-ROR1-VL and anti-ROR1-VH domains, having four polypeptide chains and including parental anti-ROR1-VL and anti-ROR1-VH structures Domain of TRIDENT-B, TRIDENT-C with three polypeptide chains and including optimized anti-ROR1-VL (14) and anti-ROR1-VH (8) domains and four polypeptide chains and including optimized anti-ROR1- The TRIDENT-D of the VL (14) and anti-ROR1-VH (8) domains has been discussed above. The general structure of the polypeptide chains of these three- and four-chain ROR1 x CD3 x CD8 trivalent binding molecules is provided in detail above. Specific anti-ROR1-VL and anti-ROR1-VH domains present in TRIDENT-A, TRIDENT-B, TRIDENT-C and TRIDENT-D are provided in Table 10 .

使用BIACORE®分析來研究ROR1 x CD3 x CD8三價結合分子中的每一種與ROR1的結合動力學，其中在BIACORE®分析中，基本上如上所述使每種三價結合分子(6.25至100nM)通過固定化的shROR1-His。從這些研究計算的k_a 、k_d 和K_D 顯示在表 10 中，其顯示包括優化的抗-ROR1-VL和抗-ROR1-VH結構域的ROR1 x CD3 x CD8三價結合分子具有改善的結合親和力。此外，也顯示TRIDENT-A和TRIDENT-B能夠結合ROR1和CD3兩者，證明ROR1 x CD3 x CD8三價結合分子保持雙重抗原結合能力。 BIACORE® analysis was used to study the binding kinetics of each of the ROR1 x CD3 x CD8 trivalent binding molecules to ROR1, wherein in the BIACORE® assay, each trivalent binding molecule (6.25 to 100 nM) was made essentially as described above. By immobilized shROR1-His. The k _a , k _d and K _D calculated from these studies are shown in Table 10 , which shows an improvement in the ROR1 x CD3 x CD8 trivalent binding molecule including the optimized anti-ROR1-VL and anti-ROR1-VH domains. Combine affinity. In addition, it was also shown that TRIDENT-A and TRIDENT-B were able to bind both ROR1 and CD3, demonstrating that the ROR1 x CD3 x CD8 trivalent binding molecule maintains dual antigen binding ability.

基本上如實施例 1 中所述使用LDH釋放試驗來評估雙特異性ROR1 x CD3三多肽鏈雙抗體DART-A和三特異性ROR1 x CD3 x CD8三價結合分子TRIDENT-A和TRIDENT-B介導重定向細胞殺傷的能力。對於這些研究，將DART-A、TRIDENT-A、TRIDENT-B或陰性對照(具有四條多肽鏈的三特異性結合分子，其結合不相關的抗原、CD3和CD8)與效應子全T-細胞和靶腫瘤細胞(JIMT-1乳腺癌細胞、NCI-H1975細胞、Calu-3肺腺癌細胞)一起以10:1的效應子與靶的比例一起孵育24小時。使用DART-A、TRIDENT-A和TRIDENT-B的5倍連續稀釋物。每種靶腫瘤細胞類型的代表性細胞毒性曲線示於圖 22A-22C 中，EC50值提供於表 11 中。 The LDH release assay was used essentially as described in Example 1 to assess the bispecific ROR1 x CD3 tripeptide chain diabody DART-A and the trispecific ROR1 x CD3 x CD8 trivalent binding molecules TRIDENT-A and TRIDENT-B Mediates the ability to redirect cell killing. For these studies, DART-A, TRIDENT-A, TRIDENT-B or a negative control (a trispecific binding molecule with four polypeptide chains that bind to unrelated antigens, CD3 and CD8) and effector T-cells and Target tumor cells (JIMT-1 breast cancer cells, NCI-H1975 cells, Calu-3 lung adenocarcinoma cells) were incubated together at a ratio of 10:1 effector to target for 24 hours. Five-fold serial dilutions of DART-A, TRIDENT-A and TRIDENT-B were used. Representative cytotoxicity curves for each target tumor cell type are shown in Figures 22A-22C , and EC50 values are provided in Table 11 .

這些研究表明，與具有相同的抗-ROR1-VL和抗-ROR1-VH結構域的雙特異性ROR1 x CD3雙抗體相比，三特異性ROR1 x CD3 x CD8三價結合分子具有介導對腫瘤細胞重定向細胞殺傷的更好的能力。These studies indicate that the trispecific ROR1 x CD3 x CD8 trivalent binding molecule mediates tumors compared to the bispecific ROR1 x CD3 diabody with the same anti-ROR1-VL and anti-ROR1-VH domains. Cells redirect cells for better killing ability.

本說明書提到的所有出版物和專利通過參考併入本文，達到如同具體和單獨指出每個單個出版物或專利申請通過參考以其整體併入的相同程度。儘管已經結合其具體實施方式描述了本發明，但是應當理解，其能夠被進一步修改，並且本申請旨在覆蓋大體上根據本發明原理並且包括與本公開的偏離的本發明的任何變型、用途或改變，只要在本發明所屬領域的已知或習慣實踐內並且如可應用至本文之前所闡釋的本質特徵。All publications and patents mentioned in this specification are hereby incorporated by reference in their entirety in their entirety in the extent the same Although the present invention has been described in connection with the specific embodiments thereof, it is understood that the subject matter of the invention The changes are as long as they are within the known or customary practice of the art to which the invention pertains and as applied before the application.

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圖 1 提供了由兩條多肽鏈組成的、具有兩個表位-結合位點的代表性共價結合的雙抗體的示意圖，每條多肽鏈均具有E-螺旋或K-螺旋異源二聚體-促進結構域(下文提供了可選擇的的異源二聚體-促進結構域)。半胱氨酸殘基可以存在於連接體和/或存在於異源二聚體-促進結構域中，如圖 3B 所示。以相同的陰影或填充式樣顯示識別相同表位元的VL和VH結構域。圖 2 提供了由兩條多肽鏈組成的、具有兩個表位-結合位點的代表性共價結合的雙抗體分子的示意圖，每條多肽鏈均具有CH2和CH3結構域，以便締合的鏈形成Fc區域的全部或部分。以相同的陰影或填充式樣顯示識別相同表位元的VL和VH結構域。圖 3A-3C 提供了由兩對多肽鏈(即，總共四條多肽鏈)組成的、具有四個表位-結合位點的代表性共價結合的四價雙抗體的示意圖。每對的一條多肽均具有CH2和CH3結構域，以便締合的鏈形成Fc區域的全部或部分。以相同的陰影或填充式樣顯示識別相同表位元的VL和VH結構域。兩對多肽鏈可以是相同的。在其中這兩對多肽鏈相同並且VL和VH結構域識別不同的表位(如圖 3A-3B 所示)這樣的實施方案中，所得分子具有四個表位-結合位點，並且對於每個結合表位是雙特異性和二價的。在其中VL和VH結構域識別相同的表位(例如，相同的VL結構域CDR和相同的VH結構域CDR在兩條鏈上都被使用)這樣的實施方案中，所得分子具有四個表位-結合位點，並且對於單表位是單特異性和四價的。可選地，兩對多肽鏈可以是不同的。在其中這兩對多肽鏈是不同的並且每對多肽的VL和VH結構域識別不同的表位(如圖 3C 中不同的陰影和式樣所示)這樣的實施方案中，所得分子具有四個表位-結合位點並且對於每個結合表位是四特異性和單價的。圖 3A 顯示含有Fc區域的雙抗體，其含有包括半胱氨酸殘基的肽異源二聚體-促進結構域。圖 3B 顯示含有Fc區域的雙抗體，其含有包括半胱氨酸殘基和連接體(具有任選的半胱氨酸殘基)的E-螺旋和K-螺旋異源二聚體-促進結構域。圖 3C 顯示含有Fc區域的雙抗體，其包括抗體CH1和CL結構域。圖 4A 和 4B 提供了由三條多肽鏈組成的、具有兩個表位-結合位點的代表性共價結合的雙抗體分子的示意圖。多肽鏈中的兩條均具有CH2和CH3結構域，以便締合的鏈形成Fc區域的全部或部分。包括VL和VH結構域的多肽鏈進一步包括異源二聚體-促進結構域。以相同的陰影或填充式樣顯示識別相同表位元的VL和VH結構域。圖 5 提供了由五條多肽鏈組成的、具有四個表位-結合位點的代表性共價結合的雙抗體分子的示意圖。多肽鏈中的兩條均具有CH2和CH3結構域，以便締合的鏈形成包括Fc區域的全部或部分的Fc區域。包括連接的VL和VH結構域的多肽鏈進一步包括異源二聚體-促進結構域。以相同的陰影或填充式樣顯示識別相同表位元的VL和VH結構域。圖 6A-6F 提供了具有三個表位-結合位元點的含有Fc區域的代表性三價結合分子的示意圖。圖 6A 和 6B 分別示意性地示出了包括兩個雙抗體型(diabody-type)結合結構域和具有不同結構域取向的Fab型(Fab-type)結合結構域的三價結合分子的結構域，其中雙抗體型結合結構域在Fc區域的N-末端或C-末端。圖 6A 和 6B 中的分子包括四條鏈。圖 6C 和 6D 分別示意性地示出了包括在Fc區域的N-末端的兩個雙抗體型結合結構域和Fab型結合結構域或scFv型結合結構域的三價結合分子的結構域，其中在Fab型結合結構域中，輕鏈和重鏈通過多肽間隔體連接。圖 6E 和 6F 中的三價結合分子分別示意性地示出了包括在Fc區域的C-末端的兩個雙抗體型結合結構域和Fab型結合結構域或scFv型結合結構域的三價結合分子的結構域，其中在Fab型結合結構域中，輕鏈和重鏈通過多肽間隔體連接。圖 6C-6F 中的三價結合分子包括三條鏈。以相同的陰影或填充式樣顯示識別相同表位元的VL和VH結構域。圖 7A-7B 描繪了親本ROR1-結合分子的非優化的抗-ROR1-VL結構域(圖 7A ， SEQ ID NO:6 )和非優化的VH結構域(圖 7B ， SEQ ID NO:7 )的氨基酸序列。底線表示CDR殘基，框表示在優選的優化的抗-ROR1-結合分子的序列中突變的殘基；Kabat位置用箭頭並且通過序列下方的編號指示，連續的氨基酸殘基編號在序列上方指示。圖 8A-8B 顯示了ROR1 x CD3雙特異性雙鏈共價結合的雙抗體：DART-1、DART-2、DART-16和DART-20介導對JIMT-1乳腺癌細胞的重定向細胞殺傷的能力，如通過細胞相關的螢光素酶活性(圖 8A )或在細胞裂解後乳酸脫氫酶(LDH)向培養基中的釋放所測量的(圖 8B )。圖 9A-9B 顯示了ROR1 x CD3雙特異性雙鏈共價結合的雙抗體DART-1、DART-14、DART-15、DART-22和DART-23介導對JIMT-1乳腺癌細胞的重定向細胞殺傷的能力，如通過細胞相關的螢光素酶活性(圖 9A )或在細胞裂解後乳酸脫氫酶(LDH)向培養基中的釋放所測量的(圖 9B )。圖 10A-10C 顯示了ROR1 x CD3雙特異性雙鏈共價結合的雙抗體DART-1、DART-22和DART-25介導對JIMT-1乳腺癌細胞(圖 10A )、HBL-2套細胞淋巴瘤細胞(圖 10B )或Jeko-1套細胞淋巴瘤細胞(圖 10C )的重定向細胞殺傷的能力，如通過在細胞裂解後乳酸脫氫酶(LDH)向培養基中的釋放所測量的。圖 11A-11B 採用夾心ELISA顯示了ROR1 x CD3雙特異性雙鏈和三鏈雙抗體的雙抗原結合能力。雙鏈雙抗體DART-1和DART-A的結合曲線顯示在圖 11A 中(結合是450nm處吸光度的函數)，三鏈雙抗體DART-A、DART-B和DART-C的結合曲線的平均值顯示在圖 11B 中。圖 12A-12D 是流式細胞儀(FACS cytometry)圖譜(profile)的描繪，通過FACS顯示了ROR1 x CD3雙特異性三鏈雙抗體DART-D與表達ROR1的癌細胞系HOP-92(圖 12A )、PC-3(圖 12B )和HBL-2(圖 12C )結合的能力，以及與表達CD3的人原代T細胞(圖 12D )結合的能力。圖 13A-13D 顯示了ROR1 x CD3雙特異性雙鏈和三鏈雙抗體DART-1和DART-A介導對JIMT-1乳腺癌細胞(圖 13A )、A549肺癌細胞(圖 13B )、HBL-2套細胞淋巴瘤細胞(圖 13C )和RECA0201癌幹細胞(圖 13D )的重定向細胞殺傷的能力。通過在細胞裂解後乳酸脫氫酶(LDH)向培養基中的釋放來測量細胞毒性。圖 14A-14B 顯示了三鏈雙抗體DART-A、DART-C和DART-D介導對JIMT-1乳腺癌細胞(圖 14A )和NCI-H1975細胞(圖 14B )的重定向細胞殺傷的能力。通過在細胞裂解後乳酸脫氫酶(LDH)向培養基中的釋放來測量細胞毒性。圖 15A-15H 顯示了代表性三鏈ROR1 x CD3雙特異性雙抗體DART-D介導對HBL-2 B細胞淋巴瘤細胞(圖 15A )、HOP-92肺腺癌細胞(圖 15B )；PC-3M***癌細胞(圖 15C )；Daoy成神經管細胞瘤細胞(圖 15D )；和Saos-2骨肉瘤細胞(圖 15E )、U-2 OS骨肉瘤細胞(圖 15F )和MG-63骨肉瘤細胞(圖 15 G)的重定向細胞殺傷的能力。正如預期的，DART-D不介導對ROR1陰性CHO細胞的重定向細胞殺傷(圖 15H )。通過在細胞裂解後乳酸脫氫酶(LDH)向培養基中的釋放來測量細胞毒性。圖 16A-16B 顯示了代表性三鏈ROR1 x CD3雙特異性雙抗體DART-D在靶NCI-H1975細胞和PBMC存在下介導細胞毒性的能力(圖 16A )，在PBMCs存在下沒有觀察到細胞毒性(圖 16B )。通過在細胞裂解後乳酸脫氫酶(LDH)向培養基中的釋放來測量細胞毒性。圖 17A-17D 顯示了代表性三鏈ROR1 x CD3雙特異性雙抗體DART-D以劑量依賴的方式在表達ROR1的NCI-H1975靶細胞的存在下上調在CD4⁺ (圖 17A 和 17C )和CD8⁺ T細胞亞群(圖 17B 和 17D )上的CD69(圖 17A-17B )和CD25(圖 17C-17D )T細胞啟動標記物的能力。圖 18A-18F 顯示在PBMCs的培養上清液中的IFN-γ(圖 18A )、TNF-α(圖 18B )、IL-10(圖 18C )、IL-6(圖 18D )、IL-4(圖 18E )、IL-2(圖 18F )細胞因數水準，其中所述PBMCs在表達ROR1的靶細胞(實心符號)存在下被DART-D(實心方形)或陰性對照雙抗體(空心菱形)處理或PBMCs單獨地被DART-D(空心方形)或陰性對照雙抗體(空心圓)處理。圖 19A-19B 顯示ROR1 x CD3雙特異性雙抗體DART-1(圖 19A )和DART-A(圖 19B )相對於媒介對照在鼠共混異種移植模型中，在體內阻止或抑制HBL-2套細胞淋巴瘤細胞的腫瘤生長或發展的能力。圖 20A-20B 顯示ROR1 x CD3雙特異性雙抗體DART-A(圖 20A )和DART-D(圖 20B )相對於媒介對照在鼠PBMC重構的異種移植模型中，在體內阻止或抑制HOP-92肺腺癌細胞的腫瘤生長或發展的能力。圖 21A-21B 顯示ROR1 x CD3雙特異性雙抗體DART-B(圖 21A ) 和DART-D(圖 21B )相對於媒介對照在PBMC重構的鼠異種移植模型中，在體內阻止或抑制NCI-H1975肺癌細胞的腫瘤生長或發展的能力。圖 22 顯示ROR1 x CD3雙特異性雙抗體DART-B相對於媒介對照在共混鼠異種移植模型中，在體內阻止或抑制REC1套細胞癌細胞的腫瘤生長或發展的能力。圖 23 顯示ROR1 x CD3雙特異性雙抗體DART-D相對於媒介對照在PBMC重構的鼠異種移植模型中，在體內阻止或抑制REC1套細胞癌細胞的腫瘤生長或發展的能力。圖 24 顯示ROR1 x CD3雙特異性雙抗體DART-D相對於媒介對照在鼠共混異種移植模型中，在體內阻止或抑制DAOY促結蒂組織增生的小腦成神經管細胞瘤細胞的腫瘤生長或發展的能力。圖 25A-25C 顯示雙特異性ROR1 x CD3三鏈雙抗體DART-A和三特異性ROR1 x CD3 x CD8三價結合分子TRIDENT-A和TRIDENT-B介導對JIMT-1乳腺癌細胞(圖 25A )、NCI-H1975細胞(圖 25B )和Calu-3肺腺癌細胞(圖 25C )的重定向細胞殺傷的能力。通過在細胞裂解後乳酸脫氫酶(LDH)向培養基中的釋放來測量細胞毒性。 Figure 1 provides a schematic representation of a representative covalently bound diabody consisting of two polypeptide chains with two epitope-binding sites, each having an E-helix or K-helix heterodimerization The body-promoting domain (optional heterodimer-promoting domain is provided below). Cysteine residues in the linker may be present and / or is present in the heterodimer - promoting domains, as shown in Figure 3B. The VL and VH domains that recognize the same epitope are displayed in the same shaded or filled pattern. Figure 2 provides a schematic representation of a representative covalently bound diabody molecule consisting of two polypeptide chains with two epitope-binding sites, each having a CH2 and CH3 domain for association The strand forms all or part of the Fc region. The VL and VH domains that recognize the same epitope are displayed in the same shaded or filled pattern. Figures 3A-3C provide schematic representations of representative covalently bound tetravalent diabodies consisting of two epitope-binding sites consisting of two pairs of polypeptide chains (i.e., a total of four polypeptide chains). Each polypeptide of each pair has a CH2 and CH3 domain such that the associated strand forms all or part of the Fc region. The VL and VH domains that recognize the same epitope are displayed in the same shaded or filled pattern. The two pairs of polypeptide chains can be identical. In such an embodiment wherein two identical pairs of polypeptide chains and different VL and VH domains identified in Table (as shown in FIGS. 3A-3B) bits, the resulting molecule has four epitope - binding site, and for each Binding epitopes are bispecific and bivalent. In embodiments where the VL and VH domains recognize the same epitope (eg, the same VL domain CDRs and the same VH domain CDRs are used on both strands), the resulting molecule has four epitopes a binding site and is monospecific and tetravalent for a single epitope. Alternatively, the two pairs of polypeptide chains can be different. Such embodiment in which two polypeptide chains are different and each pair of VL and VH domains recognize different epitopes of the polypeptide (e.g., FIG. 3C shown in different shades and patterns), the resulting molecule has four table The position-binding site is tetraspecific and monovalent for each binding epitope. Figure 3A shows a diabody containing an Fc region containing a peptide heterodimer-promoting domain comprising a cysteine residue. Figure 3B shows a diabody containing an Fc region comprising an E-helix and a K-helix heterodimer-promoting structure comprising a cysteine residue and a linker (with an optional cysteine residue) area. Figure 3C shows a diabody containing an Fc region comprising the antibody CH1 and CL domains. 4A and 4B provide a three polypeptide chains, having two epitopes - a schematic representation of binding sites covalently bound diabody molecule. Both of the polypeptide chains have a CH2 and CH3 domain such that the associated strand forms all or part of the Fc region. Polypeptide chains comprising VL and VH domains further comprise a heterodimer-promoting domain. The VL and VH domains that recognize the same epitope are displayed in the same shaded or filled pattern. Figure 5 provides a schematic representation of a representative covalently bound diabody molecule consisting of five polypeptide-binding sites with four epitope-binding sites. Both of the polypeptide chains have a CH2 and CH3 domain such that the associated strand forms an Fc region comprising all or part of the Fc region. Polypeptide chains comprising linked VL and VH domains further comprise a heterodimer-promoting domain. The VL and VH domains that recognize the same epitope are displayed in the same shaded or filled pattern. Figures 6A-6F provide schematic representations of representative trivalent binding molecules containing Fc regions with three epitope-binding bit points. Figures 6A and 6B schematically illustrate the domains of a trivalent binding molecule comprising two diabody-type binding domains and a Fab-type binding domain with different domain orientations, respectively. Wherein the diabody-type binding domain is at the N-terminus or C-terminus of the Fc region. The molecules in Figures 6A and 6B comprise four chains. 6C and 6D schematically illustrates a domain comprises two diabody-type N- terminus of the Fc region and the Fab-type binding domain binding domain or binding domain scFv-type trivalent binding molecule, wherein In the Fab-type binding domain, the light and heavy chains are joined by a polypeptide spacer. The trivalent binding molecules in Figures 6E and 6F schematically illustrate the trivalent binding of two diabody-type binding domains and a Fab-type binding domain or a scFv-type binding domain, respectively, including the C-terminus of the Fc region. A domain of a molecule in which a light chain and a heavy chain are joined by a polypeptide spacer in a Fab-type binding domain. The trivalent binding molecule in Figures 6C-6F comprises three chains. The VL and VH domains that recognize the same epitope are displayed in the same shaded or filled pattern. Figures 7A-7B depict the non-optimized anti-ROR1-VL domain of the parental ROR1-binding molecule ( Figure 7A , SEQ ID NO: 6 ) and the non-optimized VH domain ( Figure 7B , SEQ ID NO: 7 ) Amino acid sequence. The bottom line represents the CDR residues and the box represents the residue mutated in the sequence of the preferred optimized anti-ROR1-binding molecule; the Kabat position is indicated by the arrow and by the number below the sequence, and the consecutive amino acid residue number is indicated above the sequence. Figures 8A-8B show ROR1 x CD3 bispecific double-strand covalently bound diabody: DART-1, DART-2, DART-16 and DART-20 mediated retinal cell killing of JIMT-1 breast cancer cells The ability to measure, for example, by cell-associated luciferase activity ( Fig. 8A ) or release of lactate dehydrogenase (LDH) into the culture medium after cell lysis ( Fig. 8B ). Figures 9A-9B show that the ROR1 x CD3 bispecific double-strand covalently bound diabody DART-1, DART-14, DART-15, DART-22 and DART-23 mediate the weight of JIMT-1 breast cancer cells The ability to direct cell killing is measured, for example, by cell-associated luciferase activity ( Fig. 9A ) or release of lactate dehydrogenase (LDH) into the culture medium after cell lysis ( Fig. 9B ). Figures 10A-10C show that the ROR1 x CD3 bispecific double-strand covalently bound diabody DART-1, DART-22 and DART-25 mediate JIM-1 breast cancer cells ( Fig. 10A ), HBL-2 sets of cells The ability of lymphoma cells ( Fig. 10B ) or Jeko-1 mantle cell lymphoma cells ( Fig. 10C ) to redirect cell killing, as measured by release of lactate dehydrogenase (LDH) into the culture medium after cell lysis. Figures 11A-11B show the dual antigen binding ability of ROR1 x CD3 bispecific double and triple chain diabody using a sandwich ELISA. The binding curves of the double-stranded diabody DART-1 and DART-A are shown in Figure 11A (the binding is a function of absorbance at 450 nm), the mean of the binding curves of the three-chain diabodies DART-A, DART-B and DART-C. Shown in Figure 11B . Figures 12A-12D are depictions of a flow cytometry (FACS cytometry) profile showing the ROR1 x CD3 bispecific triplex diabody DART-D and the ROR1-expressing cancer cell line HOP-92 by FACS ( Figure 12A) , the ability of PC-3 ( Fig. 12B ) to bind to HBL-2 ( Fig. 12C ), and the ability to bind to human primary T cells expressing CD3 ( Fig. 12D ). Figures 13A-13D show that ROR1 x CD3 bispecific and triplex diabody DART-1 and DART-A mediate JIM-1 breast cancer cells ( Fig. 13A ), A549 lung cancer cells ( Fig. 13B ), HBL- The ability of two sets of cell lymphoma cells ( Fig. 13C ) and RECA0201 cancer stem cells ( Fig. 13D ) to redirect cell killing. Cytotoxicity was measured by release of lactate dehydrogenase (LDH) into the culture medium after cell lysis. Figures 14A-14B show the ability of triple-stranded diabody DART-A, DART-C and DART-D to mediate redirected cell killing of JIMT-1 breast cancer cells ( Figure 14A ) and NCI-H1975 cells ( Figure 14B ) . Cytotoxicity was measured by release of lactate dehydrogenase (LDH) into the culture medium after cell lysis. Figures 15A-15H show representative triple-stranded ROR1 x CD3 bispecific diabody DART-D mediated to HBL-2 B cell lymphoma cells ( Fig. 15A ), HOP-92 lung adenocarcinoma cells ( Fig. 15B ); PC -3M prostate cancer cells ( Fig. 15C ); Daoy medulloblastoma cells ( Fig. 15D ); and Saos-2 osteosarcoma cells ( Fig. 15E ), U-2 OS osteosarcoma cells ( Fig. 15F ) and MG-63 flesh tumor cells (FIG. 15 G) the ability to redirect cell killing. As expected, DART-D did not mediate redirected cell killing of ROR1-negative CHO cells ( Fig. 15H ). Cytotoxicity was measured by release of lactate dehydrogenase (LDH) into the culture medium after cell lysis. Figures 16A-16B show the ability of a representative triple-stranded ROR1 x CD3 bispecific diabody DART-D to mediate cytotoxicity in the presence of target NCI-H1975 cells and PBMC ( Figure 16A ), with no cells observed in the presence of PBMCs Toxicity ( Figure 16B ). Cytotoxicity was measured by release of lactate dehydrogenase (LDH) into the culture medium after cell lysis. Figures 17A-17D show that the representative triple-stranded ROR1 x CD3 bispecific diabody DART-D is up-regulated in CD4 ⁺ ( Figures 17A and 17C ) and CD8 in the presence of NOR-H1975 target cells expressing ROR1 in a dose-dependent manner . ⁺ The ability of CD69 ( Figures 17A-17B ) and CD25 ( Figure 17C-17D ) T cells to activate markers on T cell subsets ( Figures 17B and 17D ). Figures 18A-18F show IFN-γ ( Figure 18A ), TNF-α ( Figure 18B ), IL-10 ( Figure 18C ), IL-6 ( Figure 18D ), IL-4 (B) in the culture supernatant of PBMCs. Figure 18E ), IL-2 ( Figure 18F ) cytokine levels, wherein the PBMCs are treated with DART-D (solid squares) or negative control diabody (open diamonds) in the presence of ROR1-expressing target cells (closed symbols) or PBMCs were treated individually with DART-D (open squares) or negative control diabody (open circles). Figures 19A-19B show that the ROR1 x CD3 bispecific diabodies DART-1 ( Figure 19A ) and DART-A ( Figure 19B ) prevent or inhibit HBL-2 sets in vivo in a murine blend xenograft model relative to vehicle controls. The ability of a cell lymphoma cell to grow or develop. Figures 20A-20B show that the ROR1 x CD3 bispecific diabodies DART-A ( Figure 20A ) and DART-D ( Figure 20B ) prevent or inhibit HOP- in vivo in a murine PBMC reconstituted xenograft model relative to vehicle control. 92 The ability of lung adenocarcinomas to grow or develop tumors. Figures 21A-21B show that the ROR1 x CD3 bispecific diabody DART-B ( Figure 21A ) and DART-D ( Figure 21B ) prevent or inhibit NCI in vivo in a PBMC reconstituted murine xenograft model relative to vehicle control. H1975 The ability of lung cancer cells to grow or develop tumors. Figure 22 shows the ability of the ROR1 x CD3 bispecific diabody DART-B to block or inhibit tumor growth or progression of REC1 mantle cell carcinoma cells in vivo in a blended murine xenograft model relative to vehicle control. Figure 23 shows the ability of ROR1 x CD3 bispecific diabody DART-D to block or inhibit tumor growth or progression of REC1 mantle cell carcinoma cells in vivo in a PBMC reconstituted murine xenograft model relative to vehicle control. Figure 24 shows that ROR1 x CD3 bispecific diabody DART-D is a tumor growth of cerebellar medulloblastoma cells that prevent or inhibit DAOY-promoting pedicle tissue proliferation in a murine blended xenograft model relative to vehicle control. The ability to develop. Figures 25A-25C show that the bispecific ROR1 x CD3 triplex diabody DART-A and the trispecific ROR1 x CD3 x CD8 trivalent binding molecules TRIDENT-A and TRIDENT-B mediate JIM-1 breast cancer cells ( Fig. 25A) ), NCI-H1975 cells ( Fig. 25B ) and Calu-3 lung adenocarcinoma cells ( Fig. 25C ) have the ability to redirect cell killing. Cytotoxicity was measured by release of lactate dehydrogenase (LDH) into the culture medium after cell lysis.

Claims

一種ROR1-結合分子，其包括可變輕鏈(VL)結構域和可變重鏈(VH)結構域，其中所述VL結構域具有SEQ ID NO ： 8 的氨基酸序列： QLVLTQSPSASASLGX₁ SVX₂ LTCTLSSGHKTDTIDWYQQQPGKAPRYLMX₃ LEGSGSYNKGSGVPDRFX₄ SGX₅ SSGADX₆ YLTISSLQSEDEADYYCGTDX₇ PGNYLFGGGTQLTVLG 其中X₆ 是W，並且其中： (a)X₁ 是S或G，X₂ 是K、I或N，X₃ 是K或N，X₄ 是G或不存在，X₅ 是S或I，X₇ 是Y或N； (b)X₁ 是S，X₂ 是K，X₃ 是K，X₄ 是G或不存在，X₅ 是S，並且X₇ 是N； (c)X₁ 是S，X₂ 是K，X₃ 是K，X₄ 是G或不存在，X₅ 是I，並且X₇ 是Y； (d)X₁ 是S，X₂ 是K，X₃ 是K，X₄ 是G或不存在，X₅ 是I，並且X₇ 是N；或 (e)X₁ 是S，X₂ 是K，X₃ 是K，X₄ 是G或不存在，X₅ 是S，並且X₇ 是Y。A ROR1-binding molecule comprising a variable light (VL) domain and a variable heavy (VH) domain, wherein the VL domain has the amino acid sequence of SEQ ID NO : 8 : QLVLTQSPSASASLG X ₁ SV X ₂ _{_{LTCTLSSGHKTDTIDWYQQQPGKAPRYLM X 3 LEGSGSYNKGSGVPDRF X 4 SG X}} 5 SSGAD X 6 YLTISSLQSEDEADYYCGTD X 7 PGNYLFGGGTQLTVLG wherein X ₆ is W, and wherein: (a) X ₁ is S or G, X ₂ is K, I or N, X ₃ is K or N X ₄ is G or absent, X ₅ is S or I, X ₇ is Y or N; (b) X ₁ is S, X ₂ is K, X ₃ is K, X ₄ is G or non-existent, X ₅ is S, and X ₇ is N; (c) X ₁ is S, X ₂ is K, X ₃ is K, X ₄ is G or absent, X ₅ is I, and X ₇ is Y; (d) X ₁ is S, X ₂ is K, X ₃ is K, X ₄ is G or absent, X ₅ is I, and X ₇ is N; or (e) X ₁ is S, X ₂ is K, X ₃ Is K, X ₄ is G or absent, X ₅ is S, and X ₇ is Y.

如請求項1所述的ROR1-結合分子，其中所述VH結構域包括SEQ ID NO:9的氨基酸序列： QEQLVESGGGLVQPGGSLRLSCAASGFTFS DYYMS WX₁ RQAPGKGLEWVAT IYPSSGKTYYADSX₂ KG RX₃ TISSDNAKX₄ SLYLQMNSLRAEDTAVYYCX₅ R DSYADDAALFDI WGQGTTVTVSS 其中： (a)X₁ 是V或I，X₂ 是V或A，X₃ 是L，X₄ 是N、D或Y，並且X₅ 是A或T； (b)X₁ 是V或I，X₂ 是V或A，X₃ 是F或L，X₄ 是D或Y，並且X₅ 是A或T； (c)X₁ 是V或I，X₂ 是V或A，X₃ 是F或L，X₄ 是N、D或Y，並且X₅ 是T； (d)X₁ 是V或I，X₂ 是V或A，X₃ 是L，X₄ 是N，並且X₅ 是A； (e)X₁ 是V或I，X₂ 是V或A，X₃ 是F，X₄ 是D，並且X₅ 是A； (f)X₁ 是V或I，X₂ 是V或A，X₃ 是F，X₄ 是N，並且X₅ 是T； (g)X₁ 是V或I，X₂ 是V或A，X₃ 是L，X₄ 是D，並且X₅ 是T； (h)X₁ 是I，X₂ 是A，X₃ 是F或L，X₄ 是N、D或Y，並且X₅ 是A或T； (i)X₁ 是I，X₂ 是A，X₃ 是F，X₄ 是N，並且X₅ 是A； (j)X₁ 是I，X₂ 是A，X₃ 是L，X₄ 是N，並且X₅ 是A； (k)X₁ 是I，X₂ 是A，X₃ 是F，X₄ 是D，並且X₅ 是A； (l)X₁ 是I，X₂ 是A，X₃ 是F，X₄ 是N，並且X₅ 是T；或 (m)X₁ 是I，X₂ 是A，X₃ 是L，X₄ 是D，並且X₅ 是T。The ROR1-binding molecule of claim 1, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 9: QEQLVESGGGLVQPGGSLRLSCAASGFTFS DYYMS W X ₁ RQAPGKGLEWVAT IYPSSGKTYYADSX ₂ KG R X ₃ TISSDNAK X ₄ SLYLQMNSLRAEDTAVYYC X ₅ R DSYADDAALFDI WGQGTTVTVSS : (a) X ₁ is V or I, X ₂ is V or A, X ₃ is L, X ₄ is N, D or Y, and X ₅ is A or T; (b) X ₁ is V or I, X ₂ is V or A, X ₃ is F or L, X ₄ is D or Y, and X ₅ is A or T; (c) X ₁ is V or I, X ₂ is V or A, and X ₃ is F Or L, X ₄ is N, D or Y, and X ₅ is T; (d) X ₁ is V or I, X ₂ is V or A, X ₃ is L, X ₄ is N, and X ₅ is A (e) X ₁ is V or I, X ₂ is V or A, X ₃ is F, X ₄ is D, and X ₅ is A; (f) X ₁ is V or I, and X ₂ is V or A X ₃ is F, X ₄ is N, and X ₅ is T; (g) X ₁ is V or I, X ₂ is V or A, X ₃ is L, X ₄ is D, and X ₅ is T; (h) X ₁ is I, X ₂ is A, X ₃ is F or L, X ₄ is N, D or Y, and X ₅ is A or T; (i) X ₁ is I, X ₂ is A, X ₃ is F, X ₄ is N, and X ₅ is A; (j) X ₁ is I, X ₂ is A, X ₃ is L, X ₄ is N, and X ₅ is A; (k) X ₁ is I, X ₂ is A, X ₃ is F, X ₄ is D, and X ₅ is A; (l) X ₁ is I, X ₂ is A, and X ₃ is F X ₄ is N and X ₅ is T; or (m) X ₁ is I, X ₂ is A, X ₃ is L, X ₄ is D, and X ₅ is T.

如請求項1-2中任一項所述的ROR1-結合分子，其中： (a) 所述VL包括SEQ ID NO:11 、SEQ ID NO:20 、 SEQ ID NO:21 、SEQ ID NO:22 或SEQ ID NO:23 的氨基酸序列；並且 (b) 所述VH包括SEQ ID NO:26 、 SEQ ID NO:24 、 SEQ ID NO:25 、 SEQ ID NO:26 、 SEQ ID NO:30 、SEQ ID NO:31 或SEQ ID NO:32 的氨基酸序列。The ROR1-binding molecule of any one of claims 1-2, wherein: (a) the VL comprises SEQ ID NO: 11 , SEQ ID NO: 20 , SEQ ID NO: 21 , SEQ ID NO: 22. Or the amino acid sequence of SEQ ID NO: 23 ; and (b) the VH comprises SEQ ID NO: 26 , SEQ ID NO: 24 , SEQ ID NO: 25 , SEQ ID NO: 26 , SEQ ID NO: 30 , SEQ ID NO: 31 or the amino acid sequence of SEQ ID NO:32 .

如請求項1-3中任一項所述的ROR1-結合分子，其中所述分子是抗體或其抗原結合片段。The ROR1-binding molecule of any of claims 1-3, wherein the molecule is an antibody or antigen-binding fragment thereof.

如請求項1-3中任一項所述的ROR1-結合分子，其中所述分子是： (a) 雙特異性抗體；或 (b) 雙抗體，所述雙抗體是包括兩條、三條、四條或五條多肽鏈的共價結合的複合物；或 (c) 三價結合分子，所述三價結合分子是包括三條、四條、五條或更多條多肽鏈的共價結合的複合物。The ROR1-binding molecule of any one of claims 1 to 3, wherein the molecule is: (a) a bispecific antibody; or (b) a diabodies comprising two or three, a covalently bound complex of four or five polypeptide chains; or (c) a trivalent binding molecule that is a covalently bonded complex comprising three, four, five or more polypeptide chains.

如請求項1-5中任一項所述的ROR1-結合分子，其中所述分子包括Fc區域。The ROR1-binding molecule of any one of claims 1 to 5, wherein the molecule comprises an Fc region.

如請求項5所述的ROR1-結合分子，其中所述分子是雙抗體，並且包括白蛋白-結合結構域(ABD)。The ROR1-binding molecule of claim 5, wherein the molecule is a diabody and comprises an albumin-binding domain (ABD).

如請求項6所述的ROR1-結合分子，其中所述Fc區域是變異Fc區域，所述變異Fc區域包括： (a) 降低變異Fc區域對FcγR的親和力的一個或多個氨基酸修飾；和/或 (b) 提高變異Fc區域的血清半衰期的一個或多個氨基酸修飾。The ROR1-binding molecule of claim 6, wherein the Fc region is a variant Fc region comprising: (a) one or more amino acid modifications that reduce the affinity of the variant Fc region for FcγR; and/ Or (b) one or more amino acid modifications that increase the serum half-life of the variant Fc region.

如請求項8所述的ROR1-結合分子，其中降低變異Fc區域對FcγR的親和力的所述修飾包括以下的取代：L234A；L235A；或L234A和L235A，其中所述編號是如Kabat中的EU索引的編號。The ROR1-binding molecule of claim 8, wherein the modification that reduces the affinity of the variant Fc region for FcγR comprises the substitutions: L234A; L235A; or L234A and L235A, wherein the number is an EU index as in Kabat The number.

如請求項8或9所述的ROR1-結合分子，其中提高變異Fc區域的血清半衰期的所述修飾包括以下的取代：M252Y；M252Y和S254T；M252Y和T256E；M252Y、S254T和T256E；或K288D和H435K，其中所述編號是如Kabat中的EU索引的編號。The ROR1-binding molecule of claim 8 or 9, wherein the modification that increases the serum half-life of the variant Fc region comprises the following substitutions: M252Y; M252Y and S254T; M252Y and T256E; M252Y, S254T and T256E; or K288D and H435K, where the number is the number of the EU index as in Kabat.

如請求項1-10中任一項所述的ROR1-結合分子，其中所述分子是雙特異性的，並且包括能夠免疫特異性結合ROR1的表位的兩個表位-結合位點和能夠免疫特異性結合存在於效應細胞表面上的分子的表位的兩個表位-結合位點。The ROR1-binding molecule of any one of claims 1 to 10, wherein the molecule is bispecific and comprises two epitope-binding sites capable of immunospecifically binding to an epitope of ROR1 and capable of Immunologically binds to two epitope-binding sites of an epitope present on a surface of an effector cell.

如請求項1-10中任一項所述的ROR1-結合分子，其中所述分子是雙特異性的，並且包括能夠免疫特異性結合ROR1的表位的一個表位-結合位點和能夠免疫特異性結合存在於效應細胞表面上的分子的表位的一個表位-結合位點。The ROR1-binding molecule of any one of claims 1 to 10, wherein the molecule is bispecific and comprises an epitope-binding site capable of immunospecifically binding to an epitope of ROR1 and capable of immunizing An epitope-binding site that specifically binds to an epitope of a molecule present on the surface of an effector cell.

如請求項1-10中任一項所述的ROR1-結合分子，其中所述分子是三特異性的，並且包括： (a) 能夠免疫特異性結合ROR1的表位的一個表位-結合位點； (b) 能夠免疫特異性結合存在於效應細胞表面上的第一分子的表位的一個表位-結合位點；和 (c) 能夠免疫特異性結合存在於效應細胞表面上的第二分子的表位的一個表位-結合位點。The ROR1-binding molecule of any one of claims 1 to 10, wherein the molecule is trispecific and comprises: (a) an epitope-binding site capable of immunospecifically binding to an epitope of ROR1 (b) an epitope-binding site capable of immunospecifically binding to an epitope of a first molecule present on the surface of an effector cell; and (c) a second capable of immunospecifically binding to the surface of the effector cell An epitope-binding site of an epitope of a molecule.

如請求項1-3中任一項所述的ROR1-結合分子，其中所述分子能夠同時結合ROR1和存在於效應細胞表面上的分子。The ROR1-binding molecule of any one of claims 1 to 3, wherein the molecule is capable of binding both ROR1 and a molecule present on the surface of an effector cell.

如請求項11-13中任一項所述的ROR1-結合分子，其中存在於效應細胞表面上的所述分子是CD2、CD3、CD8、TCR或NKG2D。The ROR1-binding molecule of any one of claims 11-13, wherein the molecule present on the surface of an effector cell is CD2, CD3, CD8, TCR or NKG2D.

如請求項11-15中任一項所述的ROR1-結合分子，其中所述效應細胞是細胞毒性T細胞或自然殺傷(NK)細胞。The ROR1-binding molecule of any one of claims 1 to 15, wherein the effector cell is a cytotoxic T cell or a natural killer (NK) cell.

如請求項11-16中任一項所述的ROR1-結合分子，其中存在於效應細胞表面上的所述分子是CD3。The ROR1-binding molecule of any one of claims 11-16, wherein the molecule present on the surface of the effector cell is CD3.

如請求項13所述的ROR1-結合分子，其中存在於效應細胞表面上的所述第一分子是CD3，並且存在於效應細胞表面上的所述第二分子是CD8。The ROR1-binding molecule according to claim 13, wherein the first molecule present on the surface of the effector cell is CD3, and the second molecule present on the surface of the effector cell is CD8.

如請求項11-18中任一項所述的ROR1-結合分子，其中所述分子介導表達ROR1的細胞和細胞毒性T細胞的協調結合。The ROR1-binding molecule of any one of claims 1 to 18, wherein the molecule mediates coordinated binding of cells expressing ROR1 and cytotoxic T cells.

如請求項15或17-19中任一項所述的ROR1-結合分子，其中所述分子包括： (a) CD3 mAb 1 (SEQ ID NO:75 )的VL結構域，或所述VL結構域的一個或多個CDR；和/或 (b) CD3 mAb 1 (SEQ ID NO:76 )的VH結構域或CD3 mAb 1 (D65G) (SEQ ID NO:77 )的VH結構域，或所述VH結構域的一個或多個CDR。The ROR1-binding molecule of any one of claims 15 or 17-19, wherein the molecule comprises: (a) a VL domain of CD3 mAb 1 ( SEQ ID NO: 75 ), or the VL domain One or more CDRs; and/or (b) the VH domain of CD3 mAb 1 ( SEQ ID NO: 76 ) or the VH domain of CD3 mAb 1 (D65G) ( SEQ ID NO: 77 ), or the VH One or more CDRs of a domain.

如請求項1-5、6、8-9、12、14-17和19-20中任一項所述的ROR1-結合分子，其中所述分子包括第一多肽鏈、第二多肽鏈和第三多肽鏈，並且其中： (a) 所述第一多肽鏈包括SEQ ID NO:98 、 SEQ ID NO:101 或 SEQ ID NO:102 ； (b) 所述第二多肽鏈包括SEQ ID NO:99 、 SEQ ID NO:103 或 SEQ ID NO:104 ；和 (c) 所述第三多肽鏈包括SEQ ID NO:100 。The ROR1-binding molecule of any one of claims 1-5, 6, 8-9, 12, 14-17, and 19-20, wherein the molecule comprises a first polypeptide chain, a second polypeptide chain And a third polypeptide chain, and wherein: (a) said first polypeptide chain comprises SEQ ID NO: 98 , SEQ ID NO: 101 or SEQ ID NO: 102 ; (b) said second polypeptide chain comprises SEQ ID NO: 99 , SEQ ID NO: 103 or SEQ ID NO: 104 ; and (c) the third polypeptide chain comprises SEQ ID NO: 100 .

一種藥物組合物，其包括有效量的如請求項1-21中任一項所述的ROR1-結合分子和藥學上可接受的載體、賦形劑或稀釋劑。A pharmaceutical composition comprising an effective amount of the ROR1-binding molecule of any one of claims 1 to 21 and a pharmaceutically acceptable carrier, excipient or diluent.

如請求項1-21中任一項所述的ROR1-結合分子或如請求項22所述的藥物組合物在治療與ROR1表達相關或特徵在於ROR1表達的疾病或病症中的用途。The use of the ROR1-binding molecule of any one of claims 1 to 21, or the pharmaceutical composition of claim 22, for the treatment of a disease or condition associated with or characterized by ROR1 expression.

如請求項23所述的用途，其中與ROR1表達相關或特徵在於ROR1表達的所述疾病或病症是癌症。The use of claim 23, wherein the disease or condition associated with or characterized by ROR1 expression is cancer.

如請求項23所述的用途，其中所述癌症的特徵在於存在選自以下細胞的癌細胞：腎上腺腫瘤、AIDS相關的癌症、軟組織腺泡狀肉瘤、星形細胞瘤、腎上腺癌、膀胱癌、骨癌、腦和脊髓癌、轉移性腦腫瘤、B細胞癌、乳腺癌、頸動脈體瘤、宮頸癌、軟骨肉瘤、脊索瘤、嫌色細胞腎細胞癌、透明細胞癌、結腸癌、結直腸癌、皮膚良性纖維組織細胞瘤、促結締組織增生小圓細胞瘤、室管膜細胞瘤、尤文氏瘤、骨外黏液樣軟骨肉瘤、不完全性骨纖維生成、骨的纖維發育異常、膽囊癌或膽管癌、胃癌、妊娠滋養層疾病、生殖細胞瘤、頭頸癌、肝細胞癌、胰島細胞腫瘤、卡波西氏肉瘤、腎癌、白血病、脂肪肉瘤/惡性的脂肪瘤、肝癌、淋巴瘤、肺癌、成神經管細胞瘤、黑素瘤、腦膜瘤、多發性內分泌瘤、多發性骨髓瘤、骨髓增生異常綜合征、成神經細胞瘤、神經內分泌腫瘤、卵巢癌、胰腺癌、甲狀腺乳頭狀癌、甲狀旁腺腫瘤、兒科癌症、周圍神經鞘瘤、嗜鉻細胞瘤、垂體瘤、***癌、眼色素層後黑素瘤、罕見血液學病症、腎轉移性癌症、橫紋肌樣瘤、橫紋肌肉瘤、肉瘤、皮膚癌、軟組織肉瘤、鱗狀細胞癌、胃癌、滑膜肉瘤、睾丸癌、胸腺癌、胸腺瘤、甲狀腺轉移性癌和子宮癌。The use according to claim 23, wherein the cancer is characterized by the presence of a cancer cell selected from the group consisting of an adrenal tumor, an AIDS-related cancer, a soft tissue acinar sarcoma, an astrocytoma, an adrenal cancer, a bladder cancer, Bone cancer, brain and spinal cord cancer, metastatic brain tumor, B cell carcinoma, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, clear cell carcinoma, colon cancer, colorectal Carcinoma, benign fibrous histiocytoma of the skin, small round cell tumor of connective tissue hyperplasia, ependymoma, Ewing's tumor, extramucosal chondrosarcoma, incomplete bone fiber formation, fibrous dysplasia of the bone, gallbladder carcinoma Or cholangiocarcinoma, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, islet cell tumor, Kaposi's sarcoma, kidney cancer, leukemia, liposarcoma/malignant lipoma, liver cancer, lymphoma, Lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, intraneuropathy Tumor, ovarian cancer, pancreatic cancer, papillary thyroid carcinoma, parathyroid tumor, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, melanoma after uveal layer, rare hematology Disease, renal metastatic cancer, rhabdomyosarcoma, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, metastatic thyroid cancer, and uterine cancer.

如請求項24所述的用途，其中所述癌症選自：腎上腺癌、膀胱癌、乳腺癌、結直腸癌、胃癌、惡性膠質瘤、腎癌、非小細胞肺癌、急性淋巴細胞白血病、急性骨髓性白血病、慢性淋巴細胞白血病、慢性骨髓性白血病、毛細胞白血病、伯基特淋巴瘤、彌散性大B細胞淋巴瘤、濾泡淋巴瘤、套細胞淋巴瘤、邊緣區淋巴瘤、非霍奇金淋巴瘤、小淋巴細胞淋巴瘤、多發性骨髓瘤、黑素瘤、卵巢癌、胰腺癌、***癌、皮膚癌、腎細胞癌、睾丸癌和子宮癌。The use according to claim 24, wherein the cancer is selected from the group consisting of: adrenal cancer, bladder cancer, breast cancer, colorectal cancer, gastric cancer, glioblastoma, renal cancer, non-small cell lung cancer, acute lymphocytic leukemia, acute bone marrow. Leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, non-Hodgkin Lymphoma, small lymphocytic lymphoma, multiple myeloma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, renal cell carcinoma, testicular cancer, and uterine cancer.